Chemoenzymatic synthesis of heparin and heparan sulfate analogs

ABSTRACT

The present invention provides a one-pot multi-enzyme method for preparing UDP-sugars from simple sugar starting materials. The invention also provides a one-pot multi-enzyme method for preparing oligosaccharides from simple sugar starting materials.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation-in-part of InternationalPatent Application No. PCT/US12/47875, filed Jul. 23, 2012, which claimspriority to U.S. Provisional Patent Application No. 61/510,125, filedJul. 21, 2011, and further claims priority to U.S. Provisional PatentApplication No. 61/926,088, filed Jan. 10, 2014, U.S. Provisional PatentApplication No. 61/836,067, filed Jun. 17, 2013, and U.S. ProvisionalPatent Application No. 61/815,050, filed Apr. 23, 2013, each of which isincorporated herein by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This work was supported by NIH grants R01HD065122 and R00DK078668, andNSF grants CHE-1012511 and CHE-0548235. The United States government mayhave certain rights to the invention disclosed herein.

BACKGROUND OF THE INVENTION Reference to a “Sequence Listing, ” a Table,or a Computer Program Listing Appendix Submitted as an ASCII Text File

The Sequence Listing written in file -2050-1-1.TXT, created on Feb. 27,2014, 69,632 bytes, machine format IBM-PC, MS-Windows operating system,is hereby incorporated by reference in its entirety for all purposes.

Heparin and heparan sulfate (HS) are sulfated linear polysaccharidescomposed of alternating α1-4-linked D-glucosamine (GlcNH₂) residues and1-4 linked uronic acid α-linkage for L-iduronic acid, IdoA, andβ-linkage for D-glucuronic acid, GlcA). Possible modifications are2-O-sulfation on the uronic acid residues and one or more modificationson the glucosamine residues including N-sulfation, N-acetylation,6-O-sulfation, and 3-O-sulfation. Heparin is a mixture ofpolysaccharides that can be considered as special forms of HS withhigher levels of sulfation and iduronic acid content per disacchariderepeat unit. Heparin is mostly produced by mast cells and heparansulfates are produced by different cell types in animals. They areattractive synthetic targets due to their structural complexity whichpossesses great synthetic challenges and their important roles inregulating cancer growth, blood coagulation, inflammation, assistingviral and bacterial infections, signal transduction, lipid metabolism,and cell differentiation.

Currently, more than a hundred heparan sulfate binding proteins havebeen identified, and the structure-activity relationship studies (SAS)have revealed the interaction pattern between heparan sulfate andprotein, and further directed toward discovering and designing HSmimics. Heparin pentasaccharide sequence H₅ (also call DEFGH)GlcNS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S is essential for antithrombin IIIbinding and thrombin inhibition activities. Based on the DEFGHstructure, a new potential antithrombotic, idraparinux, was synthesizedby replacing N-sulfate groups in all three glucosamine residues ofheparin pentasaccharide DEFGH with O-sulfates and introducing methylethers at the available free hydroxyl groups and showed betteranticoagulation activity and longer duration of action than DEFGH.Another pentasaccharide sequence HexA-GlcNS-HexA-GlcNS-IdoA2S has highaffinity selectively for FGF-2 (fibroblast grow factor 2), whiletrisaccharide motif IdoA2S-GlcNS6S-IdoA2S is specific for FGF-1. N-,2-O- and 6-O-sulfations of the glucosamine residues in HS have beenshown to be required for FGF4 binding. Additionally, it has beensuggested that the N-acetylated glucosamine region rich in GlcA residuesdisplays structural plasticity and hence could mediate proteininteractions. However, the detailed information about sequencerequirement of HS that interact with many other proteins is currentlyunclear due to the lack of the technology of preparing a wide range ofstructurally defined HS.

Current chemical and enzymatic synthetic methods do not provideconvenient access to all possible heparin and HS oligosaccharidesequences. Chemical synthetic approaches are time-consuming and tedious.The production yields decrease dramatically with the increase of thelength of the target molecules. Obtaining defined structures longer thanoctasaccharides remains as a major challenge for chemical synthesis.HS-modifying enzymes have been used with other enzymes to prepareheparin polysaccharides and oligosaccharides with a limited range ofsulfation patterns. Due to the complex nature of HS-modifying enzymes,these types of methods do not allow the synthesis of a wide range of HSstructures.

Sialic acid-containing oligo- and poly-saccharides belong to anothergroup of sugars implicated in various biological and pathologicalprocesses. Sialyltransferases are the key enzymes that catalyze thetransfer of a sialic acid residue from cytidine 5′-monophosphate-sialicacid (CMP-sialic acid) to an acceptor to form sialic acid-containingproducts. They function in processes including cell-cell recognition,cell growth and differentiation, cancer metastasis, immunologicalregulation, as well as bacterial and viral infection. Besides beingprevalent in mammals, sialyltransferases have been found in somepathogenic bacteria. They are mainly involved in the formation of sialicacid-containing capsular polysaccharides (CPS) andlipooligo(poly)saccharides (LOS/LPS), serving as virulence factors,preventing recognition by host's immune system, and modulatinginteractions with the environment.

Cloning of sialyltransferases from various sources, including mammaliantissues, bacteria, and viruses has been reported. However, mostmammalian glycosyltransferases—including sialyltransferases—suffer fromrestricted substrate specificity and no or low expression in laboratoryE. coli systems. In comparison, bacterial glycosyltransferases have morepromiscuous substrate flexibility and are generally easier to accessusing E. coli expression systems.

What is needed is a convenient route to form complex oligosaccharideproducts from simple starting materials. Methods for conversion ofmonosaccharides and monosaccharide derivatives to chemically andbiologically important products, including those containingpost-glycosylational modifications and sialic acid moieties, are needed.Importantly, the intermediates and products should be formed in a highlyregio- and stereo-selective manner. The one-pot enzymatic methods of thepresent invention meet this and other needs.

BRIEF SUMMARY OF THE INVENTION

In a first embodiment, the invention provides a method of synthesizing aUDP-sugar. The method includes forming a reaction mixture comprising afirst sugar, a nucleotide-sugar pyrophosphorylase, and a first enzymeselected from a kinase and a dehydrogenase under conditions sufficientto form the UDP-sugar.

In a second embodiment, the invention provides a method of preparing anoligosaccharide. The method includes forming a first reaction mixturecontaining a first sugar, an acceptor sugar, a glycosyltransferase, anucleotide-sugar pyrophosphorylase, and an enzyme selected from a kinaseand a dehydrogenase. The first sugar is selected from a substituted orunsubstituted N-acetylglucosamine (2-acetamido-2-deoxy glucose, GlcNAc),a substituted or unsubstituted glucosamine (GlcNH₂), a substituted orunsubstituted glucuronic acid (GlcA), a substituted or unsubstitutediduronic acid (IdoA), and a substituted or unsubstitutedglucose-1-phosphate (Glc-1-P), and the acceptor sugar includes at leastone member selected from a substituted or unsubstitutedN-acetylglucosamine (GlcNAc), a substituted or unsubstituted glucosamine(GlcNH₂), a substituted or unsubstituted glucuronic acid (GlcA), and asubstituted or unsubstituted iduronic acid (IdoA). The reaction mixtureis formed under conditions sufficient to convert the first sugar to aUDP-sugar, and sufficient to couple the sugar in the UDP-sugar to theacceptor sugar. When the first sugar is substituted or unsubstitutedGlcNAc or GlcNH₂, the sugar in the UDP-sugar is coupled to a substitutedor unsubstituted GlcA or a substituted or unsubstituted IdoA of theacceptor sugar. When the first sugar is substituted or unsubstitutedGlc-1-P, substituted or unsubstituted GlcA, or substituted orunsubstituted IdoA, the sugar in the UDP-sugar is coupled to asubstituted or unsubstituted GlcNH₂ or a substituted or unsubstitutedGlcNAc of the acceptor sugar.

In a third embodiment, the invention provides a method of preparing asialylated oligosaccharide having at least two sialic acid moieties. Themethod includes forming a reaction mixture containing: a substratesugar; cytidine-5′-monophospho-sialic acid (CMP-sialic acid or CMP-Sia)or derivatives; and Photobacterium damselae α2-6-sialyltransferase(Pd2,6ST) under conditions sufficient to form the sialylatedoligosaccharide. The substrate sugar can be prepared using the one-potmulti-enzyme methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a sequence alignment of NahK_JCM1217 (GenBank accession no.BAF73925) (SEQ ID NO:19), NahK_ATCC55813 (SEQ ID NO:20), andNahK_ATCC15697(SEQ ID NO:21).

FIG. 2 shows the pH profiles of NahK_ATCC15697 (♦, filled diamond) andNahK_ATCC55813 (⋄, open diamond). Buffers used: MES, pH 6.0; Tris-HCl,pH 7.0-9.0; CAPS, pH 10.0-11.0.

FIG. 3 shows the effect of MgCl₂ on the activity of NahKs.

FIG. 4 shows the one-pot three-enzyme synthesis of UDP-GlcNAc andderivatives. Enzyme used: NahK_ATCC55813, an N-acetylhexosamine 1-kinasecloned from Bifidobacterium longum ATCC55813; PmGlmU, Pasteurellamultocida N-acetylglucosamine-1-phosphate uridylyltransferase; PmPpA,Pasteurella multocida inorganic pyrophosphatase.

FIG. 5 shows the chemical diversification at (A) the C-2 of glucosamineand (B) the C-6 of N-acetylglucosamine in UDP-sugar nucleotides.Reagents and conditions: a) K₂CO₃, CH₃OH, H₂O, 20° C., overnight, 98%;b) Py.SO₃, 2 M NaOH, H₂O, overnight, 86%; c) RCOCl, NaHCO₃, CH₃CN, H₂O;d) NaOMe, MeOH; e) H₂, Pd/C, MeOH, H₂O, 1 h, 96%.

FIG. 6 shows the pH profile of Bifidobacterium longum UDP-sugarpyrophosphorylase (BLUSP).

FIG. 7 shows the metal requirements of BLUSP.

FIG. 8 shows the synthesis of UDP-ManNAc from UDP-ManN₃ in 79% yield viathe formation of UDP-ManNH₂ by catalytic hydrogenation followed byacetylation.

FIG. 9 shows a one-pot, three-enzyme system for the synthesis ofUDP-monosaccharides and derivatives. Enzymes used: NahK_ATCC15697,Bifidobacterium infantis strain ATCC15697 N-acetylhexosamine 1-kinase;SpGalK, Streptococcus pneumoniae TIGR4 galactokinase; EcGalK, Echerichiacoli galactokinase; BLUSP, Bifidobacterium longum UDP-sugarpyrophosphorylase; PmPpA, Pasteurella multocida inorganicpyrophosphatase.

FIG. 10 shows the one-pot multienzyme synthesis of UDP-glucuronic acid,UDP-iduronic acid, and UDP-galacturonic acid.

FIG. 11 shows the results of the substrate specificity assay for theheparosan synthase activity of KfiA (FIG. 11A) and PmHS2 (FIG. 11B).Each reaction was performed at 37° C. in MES buffer (100 mM, pH 6.5) for30 min, 4 h or 16 h. Enzyme used: KfiA (1.08 μg/μL), PmHS2 (2.5×10⁻²μg/μL).

FIG. 12 shows the structures of the substrates tested in the substratespecificity assay for KfiA and PmHS2 in FIG. 11.

FIG. 13 shows the synthetic scheme for preparation of fluorescentlylabeled GlcA GlcAβ2AAMe.

FIG. 14 shows the synthesis of tetrasaccharidesGlcNTFAα1-4GlcAβ1-4GlcNTFAα1-4GlcAβ2AAMe (F14-2) andGlcNSα1-4GlcAβ1-4GlcNSα1-4GlcAβ2AA (F14-3) from trisaccharideGlcAβ1-4GlcNTFAα1-4GlcAβ2AAMe (F14-1).

FIG. 15 shows the synthesis of GlcA-TEG-PABA-biotin (F15-8).

FIG. 16 shows the one-pot four-enzyme synthesis of dissacharides withdifferent modification on C2 and C6. Enzymes used: NahK_ATCC55813,N-acetylhexosamine 1-kinase cloned from Bifidobacterium longumATCC55813; PmGlmU, Pasteurella multocida N-acetylglucosamine-1-phosphateuri-dylyltransferase; PmPpA, Pasteurella multocida inorganicpyrophosphatase; PmHS2, Pasteurella multocida heparosan synthase 2.

FIG. 17 shows the structures of UDP-GlcNAc derivatives F17-1-F17-12including UDP-GlcNAc (F17-1), UDP-GlcNTFA (F17-2), UDP-GlcNAc (F17-3),UDP-GlcNAcN₃ (F17-4), UDP-GlcNH₂ (F17-5), UDP-GlcN₃ (F17-6), UDP-GlcNS(F17-7), UDP-GlcNAc6N₃ (F17-8), UDP-GlcNAc6NGc (F17-9), UDP-GlcNAc6NH₂(F17-10j), UDP-GlcNAc6NAcN₃ (F17-11), and UDP-GlcNAc6S (F17-12).

FIG. 18 shows the enzymatic synthesis of the disaccharides. FIG. 18Ashows the one-pot four-enzyme system of the disaccharidesGlcNAcαl-4GlcAβ2AAMe (F18-1), GlcNTFAα1-4GlcAβ2AAMe (F18-2),GlcNAc6N₃α1-4GlcAβ2AAMe (F18-3). FIG. 18B shows the PmHS2-catalyzedsynthesis of the disaccharides GlcNGcα1-4GlcAβ2AAMe (F18-4),GlcNAcN₃α1-4GlcAβ2AAMe (F18-5), GlcNAc6NGcα1-4GlcAβ2AAMe (F18-6).

FIG. 19 shows the enzymatic synthesis of trisaccharides fromdisaccharides via in situ generation of UDP-GlcA from Glc-1-P catalyzedby Echerichia coli glucose-1-phosphate uridylyltransferase (EcGaIU),Pasteurella multocida UDP-glucose dehydrogenase (PmUgd), and PmHS2.

FIG. 20 shows the one-pot three-enzyme synthesis of trisaccharidesGlcAβ1-4GlcNAcα1-4GlcAβ2AAMe (F20-1), GlcAβ1-4GlcNTFAcα1-4GlcAβ2AAMe(F20-2), GlcAβ1-4GlcNAc6N₃α1-4GlcAβ2AAMe (F20-3),GlcAβ1-4GlcNGcα1-4GlcAβ2AAMe (F20-4), GlcAβ1-4GlcNAcN₃α1-4GlcAβ2AAMe(F20-5), GlcAβ1-4GlcNAc6NGcα1-4GlcAβ2AAMe (F20-6).

FIG. 21 shows the one-pot four-enzyme synthesis of tetrasaccharideGlcNAc6N₃α1-4GlcAβ1-4GlcNTFAα1-4GlcAβ2AAMe (F21-1) from trisaccharideGlcAβ1-4GlcNTFAα1-4GlcAβ2AAMe (F20-1).

FIG. 22 shows the synthesis of tetrasaccharidesGlcNAc6N₃α1-4GlcAβ1-4GlcNH₂α1-4GlcAβ2AA (F22-1),GlcNAc6N₃α1-4GlcAβ1-4GlcNSα1-4GlcAβ2AA (F22-2),GlcNAc6NH₂α1-4GlcAβ1-4GlcNSα1-4GlcAβ2AA (F22-3),GlcNAc6NSα1-4GlcAβ1-4GlcNSα1-4GlcAβ2AA (F22-4) fromGlcNAc6N₃α1-4GlcAβ1-4GlcNTFAα1-4GlcAβ2AAMe (F21-1) by chemicalmodifications. Reagents and conditions: (a) K₂CO₃, H₂O, r.t. overnight,81%; (b) Py.SO₃, 2 M NaOH, H₂O, 3d, 70%; (c) H₂, Pd/C, MeOH, H₂O, 1 h.

FIG. 23 shows the inhibitory activities of LMWH or compoundsF24-1-F24-16 (see FIG. 24 for structures) against the binding of humanfibroblast growth factors FGF-1 (FIG. 23A), FGF-2 (FIG. 23B), or FGF-4(FIG. 23C) to the heparin-biotin immobilized on NeutrAvidin-coated384-well plates. Samples without LMWH or monosaccharide/tetrasaccharideinhibitors were used as positive controls (P.C.).

FIG. 24 shows structures of compounds F24-1-F24-16 used in FIG. 23 forinhibition studies of the binding of human fibroblast growth factorsFGF-1, FGF-2, and FGF-4 to the heparin-biotin immobilized onNeutrAvidin-coated 384-well plates.

FIG. 25 shows thin-layer chromatography (TLC) analysis data for AtGlcAKreactions. Lanes: 1, ATP; 2, GlcA; 3, reaction with GlcA and ATP; 4,GalA; 5, reaction with GalA and ATP; 6, IdoA; 7, reaction with IdoA andATP; 8, xylose; 9; reaction with xylose and ATP. Developing solvent usedfor running TLC: n-PrOH:H₂O:NH₄OH=7:4:2 (by volume).

FIG. 26 shows LC-MS assay data for AtGlcAK-catalyzed synthesis ofsugar-1-phosphate from sugar and ATP. FIG. 26A, AtGlcAK kinase reactionusing GlcA as the starting sugar; FIG. 26B, AtGlcAK kinase reactionusing GalA as the starting sugar; FIG. 26C, AtGlcAK kinase reactionusing IdoA as the starting sugar.

FIG. 27 shows pH profiles of KfiA (FIG. 27A) and PmHS2 (FIG. 27B).Buffers used were: Na₂HPO₄/citric acid, pH 4.0; MES, pH 5.0-6.5;TrisHCl, pH 7.0-9.0; and CAPS, pH 10.0.

FIG. 28 shows metal effects on the heparosan synthase activity of KfiA(FIG. 28A) and PmHS2 (FIG. 28B).

FIG. 29 shows high-resolution mass spectrometry (Orbitrap HRMS) assayfor the synthesis of UDP-GlcNAc3N₃ from GlcNAc3N₃, ATP, and UTP usingone-pot three-enzyme reactions containing NahK, PmGlmU, and PmPpA.

FIG. 30 shows LC-MS or high resolution mass spectrometry (Orbitrap HRMS)assay for the synthesis of UDP-sugars from sugar, ATP, and UTP usingone-pot three-enzyme reactions containing AtGlcAK, BLUSP, and PmPpA.FIG. 30A, LC-MS assay and GlcA was used as the starting sugar; FIG. 30B,FIRMS assay and GalA was used as the starting sugar; FIG. 30C, HRMSassay and IdoA was used as the starting sugar.

FIG. 31 shows thin-layer chromatograph analysis of PmHS2-catalyzedreaction for the formation of GlcA-GlcNAc disaccharide derivatives.

FIG. 32 shows LC-MS analysis of PmHS2-catalyzed reaction for theformation of GlcA-GlcNAc disaccharide derivatives. FIG. 32A, GlcNAcα2AAwas used as the acceptor; FIG. 32B, GlcNAcβMU was used as the acceptor;FIG. 32C, GlcNAcαProN₃ was used as the acceptor; FIG. 32D, GlcNAcβProN₃was used as the acceptor.

FIG. 33 shows examples of substrate specificity for the GlcNAcTactivities of MBP-KfiA-His₆ (A) and His₆-PmHS2 (B). Each reaction wasperformed at 37° C. in MES buffer (100 mM, pH 6.5) for 24 h. Enzymeused: MBP-KfiA-His₆ (2.8 μg μl⁻¹), His₆-PmHS2 (1.1 μg μl⁻¹).

FIG. 34 shows the pH profile for AtGlcAK.

FIG. 35 shows the metal dependence of AtGlcAK-catalyzed glucuronic acidphosphorylation.

FIG. 36 shows the time-course profile for an AtGlcAK-catalyzedglucuronic acid phosphorylation reaction.

FIG. 37 shows a one-pot multi-enzyme reaction scheme for preparation ofcomplex oligosaccharide products.

FIG. 38 shows a synthetic route for preparation of a novel disialylatedoligosaccharide, DSLNnT.

FIG. 39 shows that DSLNnT protects neonatal rats from necrotizingenterocolitis. Ileum pathology scores (0: healthy; 4: completedestruction) are plotted for each animal in the different interventiongroups. DF: dam fed (number of rats n=18); FF: fed formula withoutadditional glycans (n=22); HMOs: fed formula that containsoligosaccharides isolated from pooled human milk (2 mg/mL, n=15); GOS:fed formula that contains galacto-oligosaccharides (2 mg/mL, n=15);DSLNnT: formula containing synthesized disialyl LNnT (300 μg/mL, n=14);3′″-sLNnT: formula containing synthesized 3′″-sialyl LNnT (300 μg/mL,n=19). Bars represent mean±standard deviation. ns: not significant;**P<0.01; ****P<0.0001.

DETAILED DESCRIPTION OF THE INVENTION I. General

The present invention provides a convenient and highly efficient one-potmultienzyme system for the synthesis of UDP-sugars and oligosaccharidesincluding heparin and heparosan sulfate (HS) analogs as well as humanmilk oligosaccharides (HMOs). Kinases or dehydrogenases,nucleotide-sugar pyrophosphorylases, and/or glycosyltransferases areused in one-pot reactions to convert monosaccharide precursors toUDP-sugars and/or oligosaccharides. Chemical diversification of theenzymatically formed UDP-sugars and oligosaccharides can be conducted toproduce more structural variations. In particular, non-sulfatedoligosaccharides can be selectively modified to prepare structurallydefined products with desired sulfation patterns. A diverse set ofenzymatic substrates can be used in the methods of the invention toprepare a wide range of useful UDP-sugars and oligosaccharides.

II. Definitions

As used herein, the term “first sugar” refers to a monosaccharidestarting material used in the methods of the invention. Themonosaccharide can be a hexose or a pentose. Hexoses include, but arenot limited to, glucose (Glc), glucosamine (2-amino-2-deoxy-glucose;GlcNH₂), N-acetylglucosamine (2-acetamido-2-deoxy-glucose; GlcNAc),galactose (Gal), galactosamine (2-amino-2-deoxy-galactose; GalNH₂),N-acetylgalactosamine (2-acetamido-2-deoxy-galactose; GalNAc), mannose(Man), mannosamine (2-amino-2-deoxy-mannose; ManNH₂),N-acetylmannosamine (2-acetamido-2-deoxy-mannose; ManNAc), glucuronicacid (GlcA), iduronic acid (IdoA), and galacturonic acid (GalA).Pentoses include, but are not limited to, ribose (Rib), xylose (Xyl),and arabinose (Arb). The sugar can be a D sugar or an L sugar. The sugarcan be unsubstituted or substituted with moieties including, but notlimited to, amino groups, azido groups, amido groups, acylamido groups,N-sulfate groups (sulfamate), and O-sulfate groups. A “second sugar” andsubsequent sugars are generally defined as for the first sugar, exceptthat they are used after the first sugar in a multi-step synthesis.

As used herein, the term “UDP-sugar” refers to a sugar containing auridine diphosphate moiety. The sugar portion of the UDP-sugar isdefined as for the “first sugar” described above. UDP-sugars include,but are not limited to UDP-Glc, UDP-GlcNAc, UDP-GlcNH₂, UDP-GlcA,UDP-IdoA, UDP-GalA, UDP-Gal, UDP-GalNAc, UDP-GalNH₂, UDP-Man,UDP-ManNAc, and UDP-ManNH₂. The UDP-sugar can be unsubstituted orsubstituted as described above.

As used herein, the term “CMP-sialic acid” refers to a sialic acidhaving a cytidine-5′-monophosphate moiety. The sialic acid moiety caninclude N- and O-substituted derivatives of neuraminic acid (i.e.,5-acetamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid, or(4S,5R,6R,7S,8R)-5-amino-4,6,7,8,9-pentahydroxy-2-oxo-nonanoic acid).CMP-sialic acids include, but are not limited to, cytidine5′-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac).

As used herein, the term “oligosaccharide” refers to a compoundcontaining at least two monosaccharides covalently linked together.Oligosaccharides include disaccharides, trisaccharides,tetrasachharides, pentasaccharides, hexasaccharides, heptasaccharides,octasaccharides, and the like. Covalent linkages generally consist ofglycosidic linkages (i.e., C—O—C bonds) formed from the hydroxyl groupsof adjacent sugars. Linkages can occur between the 1-carbon and the4-carbon of adjacent sugars (i.e., a 1-4 linkage), the 1-carbon and the3-carbon of adjacent sugars (i.e., a 1-3 linkage), the 1-carbon and the6-carbon of adjacent sugars (i.e., a 1-6 linkage), or the 1-carbon andthe 2-carbon of adjacent sugars (i.e., a 1-2 linkage). Linkages canoccur between the 2-carbon and the 3-carbon of adjacent sugars (i.e., a2-3 linkage), the 2-carbon and the 6-carbon of adjacent sugars (i.e., a2-6 linkage), the 2-carbon and the 8-carbon of adjacent sugars (i.e., a2-8 linkage), or the 2-carbon and the 9-carbon of adjacent sugars (i.e.,a 2-9 linkage). A sugar can be linked within an oligosaccharide suchthat the anomeric carbon is in the α- or β-configuration. Theoligosaccharides prepared according to the methods of the invention canalso include linkages between carbon atoms other than the 1-, 2-, 3-,4-, and 6-carbons or the 2-, 3-, 6-, 8-, and 9-carbons.

As used herein, the term “sialylated oligosaccharide” refers to anoligosaccharide as described above having at least one sialic acidmoiety covalently linked to the oligosaccharide. The sialic acid moietyis a monosaccharide subunit and can include N- and O-substitutedderivatives of neuraminic acid (i.e.,(4S,5R,6R,7S,8R)-5-amino-4,6,7,8,9-pentahydroxy-2-oxo-nonanoic acid).

As used herein, the term “enzyme” refers to a polypeptide that catalyzesthe transformation of a starting material, such as a sugar, to anintermediate or product of the one-pot reactions of the invention.Examples of enzymes include, but are not limited to, kinases,dehydrogenases, nucleotide-sugar pyrophosphorylases, pyrophosphatases,and glycosyltransferases. Other enzymes may be useful in the methods ofthe invention.

As used herein, the term “kinase” refers to a polypeptide that catalyzesthe covalent addition of a phosphate group to a substrate. The substratefor a kinase used in the methods of the invention is generally a sugaras defined above, and a phosphate group is added to the anomeric carbon(i.e. the “1” position) of the sugar. The product of the reaction is asugar-1-phosphate. Kinases include, but are not limited to,N-acetylhexosamine 1-kinases (NahKs), glucuronokinases (GlcAKs),glucokinases (GlcKs), galactokinases (GalKs), monosaccharide-1-kinases,and xylulokinases. Certain kinases utilize nucleotide triphosphates,including adenosine-5′-triphosphate (ATP) as substrates.

As used herein, the term “dehydrogenase” refers to a polypeptide thatcatalyzes the oxidation of a primary alcohol. In general, thedehyrogenases used in the methods of the invention convert thehydroxymethyl group of a hexose (i.e. the C6-OH moiety) to a carboxylicacid. Dehydrogenases useful in the methods of the invention include, butare not limited to, UDP-glucose dehydrogenases (Ugds).

As used herein, the term “nucleotide-sugar pyrophosphorylase” refers toa polypeptide that catalyzes the conversion of a sugar-1-phosphate to aUDP-sugar. In general, a uridine-5′-monophosphate moiety is transferredfrom uridine-5′-triphosphate to the sugar-1-phosphate to form theUDP-sugar. Examples of nucleotide-sugar pyrophosphorylases includeglucosamine uridylyltransferases (GlmUs) and glucose-1-phosphateuridylyltransferases (GalUs). Nucleotide-sugar pyrophosphorylases alsoinclude promiscuous UDP-sugar pyrophosphorylases, termed “USPs,” thatcan catalyze the conversion of various sugar-1-phosphates to UDP-sugarsincluding UDP-Glc, UDP-GlcNAc, UDP-GlcNH₂, UDP-Gal, UDP-GalNAc,UDP-GalNH₂, UDP-Man, UDP-ManNAc, UDP-ManNH₂, UDP-GlcA, UDP-IdoA,UDP-GalA, and their substituted analogs.

As used herein, the term “pyrophosphatase” (abbreviated as PpA) refersto a polypeptide that catalyzes the conversion of pyrophosphate (i.e.,P₂O₇ ⁴⁻, HP₂O₇ ³⁻, H₂P₂O₇ ²⁻, H₃P₂O₇) to two molar equivalents ofinorganic phosphate (i.e., PO₄ ³⁻, HPO₄ ²⁻, H₂PO₄).

As used herein, the term “glycosyltransferase” refers to a polypeptidethat catalyzes the formation of an oligosaccharide from anucleotide-sugar an acceptor sugar. Nucleotide-sugars include, but arenot limited to, nucleotide diphosphate sugars (NDP-sugars) andnucleotide monophosphate sugars (NMP-sugars) such as a cytidinemonosphophate sugar (CMP-sugar). In general, a glycosyltransferasecatalyzes the transfer of the monosaccharide moiety of an NDP-sugar orCMP-sugar to a hydroxyl group of the acceptor sugar. The covalentlinkage between the monosaccharide and the acceptor sugar can be a 1-3linkage, a 1-4 linkage, a 1-6-linkage, a 1-2 linkage, a 2-3 linkage, a2-6 linkage, a 2-8 linkage, or a 2-9 linkage as described above. Thelinkage may be in the α- or β-configuration with respect to the anomericcarbon of the monosaccharide. Other types of linkages may be formed bythe glycosyltransferases in the methods of the invention.Glycosyltransferases include, but are not limited to, heparosansynthases (HSs) glucosaminyltransferases,N-acetylglucosaminyltransferases, glucosyltransferasess,glucuronyltransferases, and sialyltransferases.

The glycosyltransferases useful in the present invention include thosein Glycosyltransferase family 80 (GT80 using CAZy nomenclature) andinclude beta-galactoside alpha-2,3-sialyltransferases that catalyze thefollowing conversion: CMP-sialic acid+β-D-galactosyl-R=CMP+α-sialicacid-(2→3)-β-D-galactosyl-R, where the acceptor is GalβOR, where R is H,a monosaccharide, an oligosaccharide, a polysaccharide, a glycopeptide,a glycoprotein, a glycolipid, or a hydroxyl-containing compound. GT80family sialyltransferases also include galactoside orN-acetylgalactosaminide alpha-2,6-sialyltransferases that catalyze thefollowing conversion: CMP-sialic acid+galactosyl/GalNAc-R=CMP+α-sialicacid-(2→6)-β-D-galactosyl/GalNAc-R, where the acceptor is GalOR orGalNAcOR, where R is H, serine or threonine on a peptide or protein, amonosaccharide, an oligosaccharide, a polysaccharide, a glycopeptide, aglycoprotein, a glycolipid, or a hydroxyl-containing compound.

As used herein, the term “CMP-sialic acid synthetase” refers to apolypeptide that catalyzes the synthesis of cytidine monophosphatesialic acid (CMP-sialic acid) from cytidine triphosphate (CTP) andsialic acid.

As used herein, the term “sialic acid aldolase” refers to an aldolasethat catalyzes a reversible reaction that converts a suitablehexosamine, hexose, pentose, or derivative (such as N-acetylmannosamine) to sialic acid via reaction with pyruvate.

As used herein, the term “substrate sugar” refers to a sugar thataccepts a sialic acid moiety from a CMP-sialic acid. The substrate sugarcan contain a monosaccharide, an oligosaccharide, or a polysaccharide.

As used herein, the term “couple” refers to catalyzing the formation ofa covalent bond between enzyme substrates. The coupling can take placevia the direct reaction of two substrates with each other.Alternatively, the coupling can include the formation of one or moreenzyme-substrate intermediates. An enzyme-substrate intermediate can, inturn, react with another substrate (or another enzyme-substrateintermediate) to form the bond between the substrates.

As used herein, the terms “treat”, “treating” and “treatment” refer toany indicia of success in the treatment or amelioration of an injury,pathology, condition, or symptom thereof; including any objective orsubjective parameter such as abatement; remission; diminishing ofsymptoms or making the symptom, injury, pathology or condition moretolerable to the subject; decreasing the duration of the symptom orcondition; or, in some situations, preventing the onset of the symptomor condition. The treatment or amelioration of symptoms can be based onany objective or subjective parameter; including, e.g., the result of aphysical examination.

As used herein, “necrotizing enterocolitis” refers to the death ofintestinal tissue in a subject.

As used herein, the term “subject” refers to a human or animal. Incertain embodiments, the subject is a human infant.

III. Mono- and Oligo-Saccharides

A number of UDP-sugars can be synthesized according to the methods ofthe invention. In general, the UDP-sugars have structures according toFormula I:

wherein each of R¹, R², and R³ is independently selected from OH, N₃,NH₂, NHSO₃ ⁻, OSO₃ ⁻, NHC(O)CH₃, NHC(O)CF₃, NHC(O)CH₂OH, andNHC(O)CH₂N₃; and R⁴ is selected from CH₂OH, CO₂ ⁻, CO₂H, CH₂N₃, CH₂NH₂,CH₂NHSO₃ ⁻, CH₂OSO₃ ⁻, CH₂NHC(O)CH₃, CH₂NHC(O)CF₃, CH₂NHC(O)CH₂OH, andCH₂NHC(O)CH₂N₃.

In some embodiments, the UDP-sugars have structures according to formulaIa:

A range of oligosaccharides can also be prepared using the methods ofthe invention. In general, the oligosaccharides contain one or more unitaccording to Formula II:

In oligosaccharide units according to Formula II, each of R^(1a),R^(1b), R^(2a), and R^(2b) is independently selected from OH, N₃, NH₂,NHSO₃ ⁻, OSO₃ ⁻, NHC(O)CH₃, NHC(O)CF₃, NHC(O)CH₂OH, or NHC(O)CH₂N₃; andeach of R^(1c) and R^(2c) is independently selected from CH₂OH, CO₂ ⁻,CO₂H, CH₂N₃, CH₂NH₂, CH₂NHSO₃ ⁻, CH₂OSO₃ ⁻, CH₂NHC(O)CH₃, CH₂NHC(O)CF₃,CH₂NHC(O)CH₂OH, or CH₂NHC(O)CH₂N₃. In some embodiments, one of R^(1c)and R^(2c) can be CO₂ ⁻ or CO₂H, while the other of R^(1c) and R^(2c)can be CH₂OH, CH₂N₃, CH₂NH₂, CH₂NHSO₃ ⁻, CH₂OSO₃ ⁻, CH₂NHC(O)CH₃,CH₂NHC(O)CF₃, CH₂NHC(O)CH₂OH, or CH₂NHC(O)CH₂N₃. R includes but not islimited to H, CH₃, CH₂CH₃, CH₂CH₂N₃, CH₂CH₂CH₂N₃, an aglycon accordingto Formula B, Formula C, Formula D, or Formula E below, substituted orunsubstituted GlcNAc, substituted or unsubstituted GlcNH₂, substitutedor unsubstituted GlcA, or substituted or unsubstituted Ido:

In some embodiments, the oligosaccharides have the structure of formulaIIa:

In some embodiments, the method provides oligosaccharides withstructures according to Formula III:

wherein each of R^(1a), R^(1b), and R^(2a) is independently selectedfrom OH, N₃, NH₂, NHSO₃ ⁻, OSO₃ ⁻, NHC(O)CH₃, NHC(O)CF₃, NHC(O)CH₂OH, orNHC(O)CH₂N₃; and R^(1c) is selected from CH₂OH, CH₂N₃, CH₂NH₂, CH₂NHSO₃⁻, CH₂OSO₃ ⁻, CH₂NHC(O)CH₃, CH₂NHC(O)CF₃, CH₂NHC(O)CH₂OH, orCH₂NHC(O)CH₂N₃.

In some embodiments, the method provides oligosaccharides withstructures according to Formula IV:

wherein each of R^(1a), R^(2a), and R^(2b) is independently selectedfrom OH, N₃, NH₂, NHSO₃ ⁻, OSO₃ ⁻, NHC(O)CH₃, NHC(O)CF₃, NHC(O)CH₂OH,and NHC(O)CH₂N₃; and R^(2c) is selected from CH₂OH, CH₂N₃, CH₂NH₂,CH₂NHSO₃ ⁻, CH₂OSO₃ ⁻, CH₂NHC(O)CH₃, CH₂NHC(O)CF₃, CH₂NHC(O)CH₂OH, orCH₂NHC(O)CH₂N₃.

In some embodiments, the present invention provides oligosaccharideshaving the structure of formula IVa:

In some embodiments, the method provides oligosaccharides withstructures according to Formula (V):

wherein each of R^(1a), R^(2a), R^(2b), and R^(3a) is independentlyselected from OH, N₃, NH₂, NHSO₃ ⁻, OSO₃ ⁻, NHC(O)CH₃, NHC(O)CF₃,NHC(O)CH₂OH, and NHC(O)CH₂N₃; and R^(2c) is selected from CH₂OH, CH₂N₃,CH₂NH₂, CH₂NHSO₃ ⁻, CH₂OSO₃ ⁻, CH₂NHC(O)CH₃, CH₂NHC(O)CF₃,CH₂NHC(O)CH₂OH, or CH₂NHC(O)CH₂N₃.

In some embodiments, the present invention provides oligosaccharideshaving a structure of formula Va:

In some embodiments, the method provides oligosaccharides withstructures according to Formula VI:

wherein each of R^(1a), R^(2a), R^(2b), R^(3a), R^(4b), and R^(4b) isindependently selected from OH, N₃, NH₂, NHSO₃ ⁻, OSO₃ ⁻, NHC(O)CH₃,NHC(O)CF₃, NHC(O)CH₂OH, or NHC(O)CH₂N₃; and each of R^(2c), R^(4c) isindependently selected from CH₂OH, CH₂N₃, CH₂NH₂, CH₂NHSO₃ ⁻, CH₂OSO₃ ⁻,CH₂NHC(O)CH₃, CH₂NHC(O)CF₃, CH₂NHC(O)CH₂OH, or CH₂NHC(O)CH₂N₃.

In some embodiments, the oligosaccharides has the structure of formulaVIa:

IV. One-Pot Method of Making UDP-Sugars

In a first embodiment, the invention provides a method of synthesizing aUDP-sugar. The method includes forming a reaction mixture comprising afirst sugar, a nucleotide-sugar pyrophosphorylase, and a first enzymeselected from a kinase and a dehydrogenase under conditions sufficientto form the UDP-sugar.

In some embodiments, the first sugar is selected from substituted orunsubstituted glucose (Glc), substituted or unsubstitutedglucose-1-phosphate (Glc-1-P), substituted or unsubstituted glucuronicacid (GlcA), substituted or unsubstituted glucuronic acid-1-phosphate(GlcA-1-P), substituted or unsubstituted iduronic acid (IdoA),substituted or unsubstituted iduronic acid-1-phosphate (IdoA-1-P),substituted or unsubstituted N-acetylglucosamine (GlcNAc), substitutedor unsubstituted N-acetylglucosamine-1-phosphate (GlcNAc-1-P),substituted or unsubstituted glucosamine (GlcNH₂), substituted orunsubstituted glucosamine-1-phosphate (GlcNH₂-1-P), substituted orunsubstituted galactose (Gal), substituted or unsubstitutedgalactose-1-phosphate (Gal-1-P), substituted or unsubstitutedgalacturonic acid (GalA), substituted or unsubstituted galacturonicacid-1-phosphate (GalA-1-P), substituted or unsubstitutedN-acetylgalactosamine (GalNAc), substituted or unsubstitutedN-acetylgalactosamine-1-phosphate (GalNAc-1-P), substituted orunsubstituted galactosamine (GalNH₂), substituted or unsubstitutedgalactosamine-1-phosphate (GalNH₂-1-P), substituted or unsubstitutedmannose (Man), substituted or unsubstituted mannose-1-phosphate(Man-1-P), substituted or unsubstituted N-acetylmannosamine (ManNAc),substituted or unsubstituted N-acetylmannosamine-1-phosphate(ManNAc-1-P), substituted or unsubstituted mannosamine (ManNH₂),substituted or unsubstituted mannosamine-1-phosphate (ManNH₂-1-P). Insome embodiments, the first sugar is selected from GlcNAc, Glc-1-P,GlcA, and IdoA.

In some embodiments, the first sugar has the formula VII:

Wherein each of R¹, R², and R³ is selected from OH, N₃, NH₂, NHSO₃ ⁻,OSO₃ ⁻, NHC(O)CH₃, NHC(O)CF₃, NHC(O)CH₂OH, and NHC(O)CH₂N₃; R⁴ isselected from CH₂OH, CO₂ ⁻, CO₂H, CH₂N₃, CH₂NH₂, CH₂NHSO₃ ⁻, CH₂OSO₃ ⁻,CH₂NHC(O)CH₃, CH₂NHC(O)CF₃, CH₂NHC(O)CH₂OH, and CH₂NHC(O)CH₂N₃; and R⁵can be H, PO₃ ²⁻, or HPO₃ ⁻. In some embodiments, the first sugar hasthe formula VIII or IX:

In general, the reaction mixture formed in the methods of the inventioncontains a nucleotide-sugar pyrophosphorylase. The nucleotide-sugarpyrophosphorylase can be, but is not limited to, a glucosamineuridyltransferase (GlmU), a Glc-1-P uridylyltransferase (GalU), or apromiscuous UDP-sugar pyrophosphorylase (USP). The present inventorshave cloned and characterized a GlmU from P. multocida (PmGlmU) that isuseful for the synthesis of UDP-sugars according to the methods of theinvention. Suitable GalUs can be obtained, for example, from yeasts suchas Saccharomyces fragilis, pigeon livers, mammalian livers such asbovine liver, Gram-positive bacteria such as Bifidobacterium bifidum,and Gram-negative bacteria such as Echerichia coli (EcGalU) (Chen X,Fang J W, Zhang J B, Liu Z Y, Shao J, Kowal P, Andreana P, and Wang P G.J. Am. chem. Soc. 2001, 123, 2081-2082). In some embodiments, thenucleotide-sugar pyrophosphorylase is a USP. USPs include, but are notlimited to, those obtained from Pisum sativum L. (PsUSP) and Arabidopsisthaliana (AtUSP), as well as enzymes obtained from protozoan parasites(such as Leishmania major and Trypanosoma cruzi) and hyperthermophilicarchaea (such as Pyrococcus furiosus DSM 3638). USPs also include humanUDP-GalNAc pyrophosphorylase AGX1, E. coli EcGlmU, and Bifidobacteriumlongum BLUSP. BLUSP was cloned and characterized by the inventors. Insome embodiments, the nucleotide-sugar pyrophosphorylase is selectedfrom AGX1, EcGlmU, EcGalU, PmGlmU, and BLUSP. In some embodiments, thenucleotide-sugar pyrophosphorylase is selected from EcGalU, PmGlmU, andBLUSP. In some embodiments, the nucleotide-sugar pyrophosphorylase isEcGalU. In some embodiments, the nucleotide-sugar pyrophosphorylase isPmGlmU. In some embodiments, the nucleotide-sugar pyrophosphorylase isBLUSP.

The reaction mixture formed in the methods of the invention alsocontains a kinase or a dehydrogenase. In some embodiments, the firstenzyme in the reaction mixture is a kinase. The kinase can be, but isnot limited to, an N-acetylhexosamine 1-kinase (NahK), a galactokinase(GalK), or a glucuronokinase (GlcAK). In some embodiments, the kinase isan NahK. The NahK can be, for example, Bifidobacterium infantisNahK_ATCC15697 or Bifidobacterium longum NahK_ATCC55813. NahK_ATCC15697and NahK_ATCC55813 were cloned and characterized by the inventors. Insome embodiments, the kinase is a GalK. The GalK can be, for example,Escherichia coli EcGalK (Chen X, Fang J W, Zhang J B, Liu Z Y, Shao J,Kowal P, Andreana P, and Wang P G. J. Am. chem. Soc. 2001, 123,2081-2082) and Streptococcus pneumoniae TIGR4 SpGalK (Chen M, Chen L L,Zou Y, Xue M, Liang M, Jing L, Guan W Y, Shen J, Wang W, Wang L, Liu J,and Wang P G. Carbohydr. Res. 2011, 346, 2421-2425).

In some embodiments, the UDP-sugar is a substituted or unsubstitutedUDP-GlcA. The first sugar employed in the synthesis of UDP-GlcA may varydepending on the enzymes that are used in the one-pot reaction. Forexample, Glc-1-P can be converted to UDP-Glc using a UDP-sugarpyrophosphorylase. UDP-GlcA can be obtained from UDP-Glc using adehydrogenase. Accordingly, the reaction mixture in some embodiments ofthe invention includes a dehydrogenase. The dehydrogenase can be, but isnot limited to, a UDP-glucose dehydrogenase (Ugd). In some embodiments,the dehydrogenase is Pasteurella multocida PmUgd. The PmUgd was clonedand characterized by the inventors. Alternatively, GlcA can be convertedto GlcA-1-P using a GlcAK. In some embodiments, therefore, the kinase inthe reaction mixture is a GlcAK. The GlcAK can be, for example,Arabidopsis thaliana AtGlcAK. The GlcA-1-P is then converted to UDP-GlcAby a UDP-sugar pyrophosphorylase such as Arabidopsis thaliana AtUSP. TheAtGlcAK was cloned and characterized by the inventors. Other sugars,including iduronic acid (IdoA) and galacturonic acid (GalA), can also beused as substrates for GlcAKs in the methods of the invention.

Various UDP-sugars can be synthesized using the methods of theinvention. In some embodiments, the UDP-sugar is selected fromsubstituted or unsubstituted UDP-Glc, substituted or unsubstitutedUDP-GlcA, substituted or unsubstituted UDP-IdoA, substituted orunsubstituted UDP-GalA, substituted or unsubstituted UDP-GlcNAc,substituted or unsubstituted UDP-GlcNH₂, substituted or unsubstitutedUDP-Gal, substituted or unsubstituted UDP-GalNAc, substituted orunsubstituted UDP-GalNH₂, substituted or unsubstituted UDP-Man,substituted or unsubstituted UDP-ManNAc, and substituted orunsubstituted UDP-ManNH₂. In some embodiments, the UDP-sugar is selectedfrom UDP-GlcNAc, UDP-GlcNH₂, UDP-GlcA, UDP-IdoA, UDP-GalA, UDP-Gal,UDP-Man, and UDP-Glc. The UDP-sugar can also have the structure offormula I described above.

The hydroxyl groups, the amino group, and the N-acetyl amino group inUDP-sugar can be substituted with any suitable substituent. In someembodiments, the hydroxyl groups, the amino group, and the N-acetylamino group in UDP-sugar can be substituted with an azide, an amine, anN-trifluoroacetyl group, an N-acyl group, an O-sulfate, or an N-sulfate.

The reaction mixture formed in the methods of the invention can furtherinclude an inorganic pyrophosphatase (PpA). PpAs can catalyze thedegradation of the pyrophosphate (PPi) that is formed during theconversion of a sugar-1-phosphate to a UDP-sugar. PPi degradation inthis manner can drive the reaction towards the formation of theUDP-sugar products. The pyrophosphatase can be, but is not limited to,Pasteurella multocida PmPpA (Lau K, Thon V, Yu H, Ding L, Chen Y,Muthana M M, Wong D, Huang R, and Chen X. Chem. Commun. 2010, 46,6066-6068).

The reaction mixture in the present methods can be formed under anyconditions sufficient to convert the first sugar to a UDP-sugar or anintermediate such as a sugar-1-phosphate. The reaction mixture caninclude, for example, buffering agents to maintain a desired pH, as wellas salts and/or detergents to adjust the solubility of the enzymes orother reaction components. In general, the reaction mixture alsoincludes one or more nucleotide triphosphates (NTPs), such as UTP orATP, that are consumed during sugar phosphorylation and UDP-sugarformation. The reaction mixture can contain a stoichiometric amount ofan NTP, with respect to the first sugar, or an excess of the NTP.Divalent metal ions, such as magnesium ions, manganese ions, cobaltions, or calcium ions, may be required to maintain the catalyticactivity of certain enzymes. Enzyme cofactors, including but not limitedto nicotinamide adenine dinucleotide (NAM, can also be included in thereaction mixture. In some embodiments, the reaction mixture furtherincludes at least one component selected from UTP, ATP, Mn²⁺, Co²⁺,Ca²⁺, and Mg²⁺. After the reaction mixture is formed, it is held underconditions that allow for the conversion of the first sugar to the UDPsugar. For example, the reaction mixture can be held at 37° C. for 1min-72 hr to form the UDP-sugar. The reaction mixture can also be heldat 25° C. to form the UDP-sugar. Other temperatures and conditions maybe suitable for forming the UDP-sugar, depending on the nature of thefirst sugar and the enzymes used for the synthesis.

In some embodiments, the invention provides a method of synthesizing aUDP-sugar of Formula I:

The method includes forming a reaction mixture comprising a first sugar,a nucleotide-sugar pyrophosphorylase, and a first enzyme selected fromthe group consisting of a kinase and a dehydrogenase under conditionssufficient to form the UDP-sugar. In some embodiments, the first sugarhas the formula VII:

Wherein each of R¹, R², and R³ is selected from OH, N₃, NH₂, NHSO₃ ⁻,OSO₃ ⁻, NHC(O)CH₃, NHC(O)CF₃, NHC(O)CH₂OH, and NHC(O)CH₂N₃; R⁴ isselected from CH₂OH, CO₂ ⁻, CO₂H, CH₂N₃, CH₂NH₂, CH₂NHSO₃ ⁻, CH₂OSO₃ ⁻,CH₂NHC(O)CH₃, CH₂NHC(O)CF₃, CH₂NHC(O)CH₂OH, and CH₂NHC(O)CH₂N₃; and R⁵can be H, PO₃ ²⁻, or HPO₃ ⁻.

Certain enzymes that are useful in the methods of the invention arecharacterized by a level of substrate promiscuity that allows for thesynthesis of various natural and non-natural UDP-sugars. The scope ofthe products can be widened further by chemically appending a range offunctionality to common enzymatically synthesized UDP-sugars. AUDP-sugar containing an azido moiety, for example, can be reduced toform an amino moiety which can be further elaborated via amide bondformation or N-sulfation to install various functional groups in theUDP-sugar. Similarly, trifluoracetamido moieties can also be convertedto amino moieties for further derivitization. Accordingly, someembodiments of the invention include converting a UDP-azido-sugar or aUDP-trifluoroacetamido-sugar to a UDP-amino-sugar. In some embodiments,the UDP amino-sugar is further converted to a UDP-acylamido-sugar or aUDP-N-sulfated-sugar.

V. One-Pot Method of Making Oligosaccharides

The method described above for preparing UDP-sugars can be extended byincorporating additional enzymes that incorporate the sugar inUDP-sugars into oligosaccharide products. Accordingly, some embodimentsof the invention provide a method of preparing an oligosaccharide. Themethod includes forming a first reaction mixture containing a firstsugar, an acceptor sugar, a glycosyltransferase, a nucleotide-sugarpyrophosphorylase, and an enzyme selected from a kinase and adehydrogenase. The first sugar is selected from a substituted orunsubstituted N-acetylglucosamine (2-acetamido-2-deoxy glucose, GlcNAc),a substituted or unsubstituted glucosamine (GlcNH₂), a substituted orunsubstituted glucuronic acid (GlcA), a substituted or unsubstitutediduronic acid (IdoA), and a substituted or unsubstitutedglucose-1-phosphate (Glc-1-P), and the acceptor sugar includes at leastone member selected from a substituted or unsubstitutedN-acetylglucosamine (GlcNAc), a substituted or unsubstituted glucosamine(GlcNH₂), a substituted or unsubstituted glucuronic acid (GlcA), and asubstituted or unsubstituted iduronic acid (IdoA). The reaction mixtureis formed under conditions sufficient to convert the first sugar to aUDP-sugar, and sufficient to couple the sugar in the UDP-sugar to theacceptor sugar. When the first sugar is substituted or unsubstitutedGlcNAc or GlcNH₂, the sugar in the UDP-sugar is coupled to a substitutedor unsubstituted GlcA or a substituted or unsubstituted IdoA of theacceptor sugar. When the first sugar is substituted or unsubstitutedGlc-1-P, substituted or unsubstituted GlcA, or substituted orunsubstituted IdoA, the sugar in the UDP-sugar is coupled to asubstituted or unsubstituted GlcNH₂ or a substituted or unsubstitutedGlcNAc of the acceptor sugar.

In some embodiments, the first sugar has the formula:

Wherein each of R¹, R², and R³ is selected from OH, N₃, NH₂, NHSO₃ ⁻,OSO₃ ⁻, NHC(O)CH₃, NHC(O)CF₃, NHC(O)CH₂OH, and NHC(O)CH₂N₃; R⁴ isselected from CH₂OH, CO₂, CO₂H, CH₂N₃, CH₂NH₂, CH₂NHSO₃ ⁻, CH₂OSO₃ ⁻,CH₂NHC(O)CH₃, CH₂NHC(O)CF₃, CH₂NHC(O)CH₂OH, and CH₂NHC(O)CH₂N₃; and R⁵can be H, PO₃ ²⁻, or HPO₃ ⁻. In some embodiments, the first sugar hasthe formula VIII or IX:

The first sugar is converted to the UDP-sugar by the UDP-sugarpyrophosphorylase and the kinase/dehydrogenase as described above. Insome embodiments, the first sugar is selected from substituted orunsubstituted glucose (Glc), substituted or unsubstitutedglucose-1-phosphate (Glc-1-P), substituted or unsubstituted glucuronicacid (GlcA), substituted or unsubstituted iduronic acid (IdoA),substituted or unsubstituted glucuronic acid-1-phosphate (GlcA-1-P),substituted or unsubstituted iduronic acid (IdoA), substituted orunsubstituted iduronic acid-1-phosphate (IdoA-1-P), substituted orunsubstituted N-acetylglucosamine (GlcNAc), substituted or unsubstitutedN-acetylglucosamine-1-phosphate (GlcNAc-1-P), substituted orunsubstituted glucosamine (GlcNH₂), substituted or unsubstitutedglucosamine-1-phosphate (GlcNH₂-1-P), substituted or unsubstitutedgalactose (Gal), substituted or unsubstituted galactose-1-phosphate(Gal-1-P), substituted or unsubstituted galacturonic acid (GalA),substituted or unsubstituted galacturonic acid-1-phosphate (GalA-1-P),substituted or unsubstituted N-acetylgalactosamine (GalNAc), substitutedor unsubstituted N-acetylgalactosamine-1-phosphate (GalNAc-1-P),substituted or unsubstituted galactosamine (GalNH₂), substituted orunsubstituted galactosamine-1-phosphate (GalNH₂-1-P), substituted orunsubstituted mannose (Man), substituted or unsubstitutedmannose-1-phosphate (Man-1-P), substituted or unsubstitutedN-acetylmannosamine (ManNAc), substituted or unsubstitutedN-acetylmannosamine-1-phosphate (ManNAc-1-P), substituted orunsubstituted mannosamine (ManNH₂), substituted or unsubstitutedmannosamine-1-phosphate (ManNH₂-1-P). In some embodiments, the UDP-sugaris a compound of Formula I.

The sugar in the UDP-sugar is, in turn, coupled to an acceptor sugar toform an oligosaccharide product. A variety of sugars can be used as theacceptor sugar. For example, the acceptor sugar can be a monosaccharide,a disaccharide, a trisaccharide, or a tetrasaccharide. Longeroligosaccharides may also be used as the acceptor sugar in the methodsof the invention. In some embodiments, the oligosaccharide can be acompound of Formula II, III, IV, V, or VI.

In general, the sugar in a UDP-sugar is coupled to an acceptor sugar bythe glycosyltransferase in the reaction mixture. Any suitableglycosyltransferase can be used in the methods of the invention. Certainglycosyltransferases have exhibited a level of substrate promiscuitythat are particularly useful for preparing a variety of oligosaccharideproducts. Promiscuous glycosyltransferases can utilize a range ofUDP-sugars and/or a range of acceptor sugars. The glycosyltransferasecan be, for example, P. multocida PmHS1 or PmHS2. Theglycosyltransferase can also be E. coli KfiA or KfiC. Otherglycosyltransferases can also be useful in the methods of the invention.In some embodiments, the glycosyltransferase is selected from PmHS1,PmHS2 and KfiA. In some embodiments, the glycosyltransferase is selectedfrom PmHS1, PmHS2, NmLgtA, NmLgtB, PmCS, PmHAS, KfiC, and KfiA.

In general, the UDP-sugar can be formed enzymatically in the one-potreaction mixture as described above. The nucleotide-sugarpyrophosphorylase can be, but is not limited to, a glucosamineuridyltransferase (GlmU), a Glc-1-P uridylyltransferase (GalU), or apromiscuous UDP-sugar pyrophosphorylase (USP). In some embodiments, thenucleotide-sugar pyrophosphorylase is selected from AGX1, EcGlmU,EcGalU, PmGlmU, and BLUSP. In some embodiments, the nucleotide-sugarpyrophosphorylase is selected from AGX1, EcGalU, and BLUSP. In someembodiments, the nucleotide-sugar pyrophosphorylase is selected fromEcGalU, PmGlmU, and BLUSP. In some embodiments, the nucleotide-sugarpyrophosphorylase is EcGalU. In some embodiments, the nucleotide-sugarpyrophosphorylase is PmGImU. In some embodiments, the nucleotide-sugarpyrophosphorylase is BLUSP.

In some embodiments, the kinase in the reaction mixture is selected froman N-acetylhexosamine 1-kinase (NahK), a galactokinase (GalK), and aglucuronokinase (GlcAK). In some embodiments, the kinase is selectedfrom NahK_ATCC15697, NahKATCC55813, EcGalK, SpGalK, and AtGlcAK. In someembodiments, the kinase is selected from NahK_ATCC15697, NahK_ATCC55813,EcGalK, and AtGlcAK. In some embodiments, the kinase is selected fromNahK_ATCC15697, NahK_ATCC55813, and AtGlcAK. In some embodiments, thekinase is EcGa1K. In some embodiments, the kinase is NahK_ATCC15697. Insome embodiments, the kinase is NahK_ATCC55813. In some embodiments, thekinase is AtGlcAK. In some embodiments, the kinase is NahK_ATCC55813.

In some embodiments, the dehydrogenase in the reaction mixture isUDP-glucose dehydrogenase (Ugd). In some embodiments, the Ugd is PmUgd.

In some embodiments, the UDP-sugar formed in the one-pot reactionmixture is selected from substituted or unsubstituted UDP-GlcNAc,substituted or unsubstituted UDP-Glc, substituted or unsubstitutedUDP-GlcA, and substituted or unsubstituted UDP-IdoA. In someembodiments, the UDP-sugar is substituted with at least one moietyselected from an azide, an amine, an N-trifluoroacetyl group, anN-acylamido group, an O-sulfate, and an N-sulfate.

In some embodiments, the reaction mixture further contains apyrophosphatase. In some embodiments, the pyrophosphatase is PmPpA.

The reaction mixture in the present methods can be formed under anysuitable conditions sufficient to prepare an oligosaccharide. Thereaction mixture can include, for example, buffering agents to maintaina desired pH, as well as salts and/or detergents to adjust thesolubility of the enzymes or other reaction components. In general, thereaction mixture also includes one or more nucleotide triphosphates(NTPs), such as UTP or ATP, that are consumed during sugarphosphorylation and UDP-sugar formation. The reaction mixture cancontain a stoichiometric amount of an NTP, with respect to the firstsugar, or an excess of the NTP. Divalent metal ions, such as magnesiumions, manganese ions, cobalt ions, or calcium ions, may be required tomaintain the catalytic activity of certain enzymes. Enzyme cofactors,including but not limited to nicotinamide adenine dinucleotide (NAM, canalso be included in the reaction mixture. In some embodiments, thereaction mixture further includes at least one component selected fromUTP, ATP, Mn²⁺, Co²⁺, Ca²⁺, and Mg²⁺. After the reaction mixture isformed, it is held under conditions that allow for preparation of theoligosaccharide. For example, the reaction mixture can be held at 37° C.for 1 min-72 hr. The reaction mixture can also be held at 25° C. Othertemperatures and conditions may be suitable for forming theoligosaccharide, depending on the nature of the sugars and the enzymesused for the synthesis.

Heparin and heparan sulfate (HS) oligosaccharides have particularlyimportant biological, pathological, and therapeutic properties. Heparinand HS are sulfated linear polysaccharides composed of alternating α1-4linked D-glucosamine (GlcNH₂) residues and 1-4 linked uronic acidresidues (α-linkage for iduronic acid, IdoA, and β-linkage forglucuronic acid, GlcA]. In order to prepare heparin and HSoligosaccharides and their analogs, the methods of the invention can beused to prepare oligosaccharides containing alternating glucosamine anduronic acid residues. The oligosaccharides can contain, for example,alternating GlcNAc residues and GlcA residues. In some embodiments, theoligosaccharide is selected from: GlcNAc-GlcA; GlcA-GlcNAc-GlcA;GlcNAc-GlcA-GlcNAc-GlcA; GlcA-GlcNAc-GlcA-GlcNAc-GlcA;GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA;GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA;GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA;GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA; GlcA-GlcNAc;GlcNAc-GlcA-GlcNAc; GlcA-GlcNAc-GlcA-GlcNAc;GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc; GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc;GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc;GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc;GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc; andGlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc-GlcA-GlcNAc. In someembodiments, each GlcA and GlcNAc are optionally independently mono- ormulti-substituted with a moiety selected from an azide, an amine, anN-trifluoroacetyl group, an N-acyl group, and an N-sulfate.

Other oligosaccharides can also be prepared using the methods of theinvention. Oligosaccharides of arbitrary length can be prepared byrepeating the one-pot reaction methods as described above. Accordingly,some embodiments of the invention provide a method for preparing anoligosaccharide as described above, wherein the method is repeated witha second sugar in place of the first sugar and the oligosaccharide inplace of the acceptor sugar. In this manner, a variety of products canbe prepared. In some embodiments, the oligosaccharides of the presentinvention can be a compound of any of Formulas II, III, IV, V, or VI.

In some embodiments, the present invention provides a method ofpreparing an oligosaccharide of formula II:

wherein the method includes forming a first reaction mixture containinga first sugar, an acceptor sugar, a glycosyltransferase, a UDP-sugarpyrophosphorylase, and/or one enzyme selected from a kinase and adehydrogenase. The first sugar is selected from a substituted orunsubstituted N-acetylglucosamine (GlcNAc), a substituted orunsubstituted glucosamine (GlcNH₂), a substituted or unsubstitutedglucoronic acid (GlcA), a substituted or unsubstituted iduronic acid(IdoA), and a substituted or unsubstituted glucose-1-phosphate(Glc-1-P), and the acceptor sugar includes at least one member selectedfrom a substituted or unsubstituted N-acetylglucosamine (GlcNAc), asubstituted or unsubstituted glucosamine (GlcNH₂), a substituted orunsubstituted glucuronic acid (GlcA), and substituted or unsubstitutediduronic acid (IdoA). The reaction mixture is formed under conditionssufficient to convert the first sugar to a UDP-sugar having a structureof formula I:

and sufficient to couple the sugar in the UDP-sugar to the acceptorsugar. The first sugar can have a structure of the formula VII:

Each of R¹, R², R³, R^(1a), R^(1b), R^(2a), and R^(2b) is independentlyselected from OH, N₃, NH₂, NHSO₃ ⁻, OSO₃ ⁻, NHC(O)CH₃, NHC(O)CF₃,NHC(O)CH₂OH, or NHC(O)CH₂N₃; each of R⁴, R^(1c), and R^(2c) isindependently selected from CH₂OH, CO₂ ⁻, CO₂H, CH₂N₃, CH₂NH₂, CH₂NHSO₃⁻, CH₂OSO₃ ⁻, CH₂NHC(O)CH₃, CH₂NHC(O)CF₃, CH₂NHC(O)CH₂OH, orCH₂NHC(O)CH₂N₃; R includes but not is limited to H, CH₃, CH₂CH₃,CH₂CH₂N₃, CH₂CH₂CH₂N₃, an aglycon according to Formula B, Formula C,Formula D, or Formula E below, substituted or unsubstituted GlcNAc,substituted or unsubstituted GlcNH₂, substituted or unsubstituted GlcA,or substituted or unsubstituted IdoA. When R⁴ is CO₂ ⁻ or CO₂H, thenR^(2c) is CO₂ ⁻ or CO₂H, and R^(1c) is selected from CH₂OH, CH₂N₃,CH₂NH₂, CH₂NHSO₃ ⁻, CH₂OSO₃ ⁻, CH₂NHC(O)CH₃, CH₂NHC(O)CF₃,CH₂NHC(O)CH₂OH, and CH₂NHC(O)CH₂N₃. Alternatively, when R⁴ is CH₂OH,CH₂N₃, CH₂NH₂, CH₂NHSO₃ ⁻, CH₂OSO₃ ⁻, CH₂NHC(O)CH₃, CH₂NHC(O)CF₃,CH₂NHC(O)CH₂OH, or CH₂NHC(O)CH₂N₃, then R^(1c) is CO₂ or CO₂H, andR^(2c) is R⁴.

Heparin and HS generally contain varying levels of sulfated sugarresidues. Examples of sulfated sugar residues include, but are notlimited to, GlcNS, containing an N-sulfate at the 2 position ofglucosamine (GlcNH₂); GlcNS3S, containing an N-sulfate at the 2 positionand an O-sulfate at the 3 position of glucosamine (GlcNH₂); GlcNS6S,containing an N-sulfate at the 2 position and an O-sulfate at the 6position of glucosamine (GlcNH₂); GlcNS3S6S, containing an N-sulfate atthe 2 position, an O-sulfate at the 3 position, and an O-sulfate at the6 position of glucosamine (GlcNH₂); GlcNAc3S, containing an O-sulfate atthe 3 position of N-acetylglucosamine (GlcNAc); GlcNAc6S, containing anO-sulfate at the 6 position of N-acetylglucosamine (GlcNAc); GlcNAc3S6S,containing an O-sulfate at the 3 position and an O-sulfate at the 6position of N-acetylglucosamine (GlcNAc); GlcNH₂3S, containing anO-sulfate at the 3 position of glucosamine (GlcNH₂); GlcNH₂6S,containing an O-sulfate at the 6 position of glucosamine (GlcNH₂);GlcNH₂3S6S, containing an O-sulfate at the 3 position and an O-sulfateat the 6 position of glucosamine (GlcNH₂); GlcA2S, containing anO-sulfate at the 2 position of glucuronic acid (GlcA); and IdoA2S,containing an O-sulfate at the 2 position of iduronic acid (IdoA).Substrate preferences for the methods of the invention will varydepending on the specific enzymes employed in the one-pot reactions. Asdescribed above, various substituted and unsubstituted sugars can beused in the methods of the invention.

The present inventors have discovered enzymes that exhibit catalyticactivity for a number of natural and non-natural UDP-sugar and acceptorsugar substrates. The oligosaccharides that are prepared using theseenzymes can contain functional moieties that can be chemically modifiedto diversify the structure of the products. For example, azido-sugarresidues or trifluoroacetamido-sugar residues can be converted toamino-sugar residues. Azido groups and trifluoracetamos groups can bemanipulated independently using orthogonal chemical methods toselectively install desired functionality at specific sites on a givenoligosaccharide. Amine-containing oligosaccharides can be furtherelaborated to form acylamino groups and sulfamate groups. Sulfamate(i.e. N-sulfate) groups, in particular, can be installed to form heparinand HS analogs.

The inventors have discovered that certain oligosaccharides containingN-sulfate groups (where O-sulfate groups would normally be present inheparin and HS) demonstrate inhibitory activity against the binding offibroblast growth factors (FGFs) to heparin. FGFs, in turn, have a rolein regulating a number of processes including angiogenesis, cellproliferation, differentiation, morphogenesis, and wound healing. Assuch, the invention provides convenient and flexible methods forpreparation of oligosaccharides with useful biological activity.

VI. One-Pot Method of Preparing Sialylated Oligosaccharides

The methods described above for preparing oligosaccharides can befurther extended by incorporating sialyltransferases that convert theoligosaccharides into sialylated oligosaccharides. Sialyltransferasesare the key enzymes that catalyze the transfer of a sialic acid residuefrom cytidine 5′-monophosphate-sialic acid (CMP-sialic acid) to anacceptor. Resulting sialic acid-containing products have been implicatedin various biological and pathological processes, including cell-cellrecognition, cell growth and differentiation, cancer metastasis,immunological regulation, as well as bacterial and viral infection.Besides being prevalent in mammals, sialyltransferases have been foundin some pathogenic bacteria. They are mainly involved in the formationof sialic acid-containing capsular polysaccharides (CPS) andlipooligo(poly)saccharides (LOS/LPS), serving as virulence factors,preventing recognition by host's immune system, and modulatinginteractions with the environment.

Human Milk Oligosaccharides

Human milk oligosaccharides (HMOs) are a mixture of more than 100glycans which constitute the third major component of human milk. Theyhave been found to contribute significantly to the gut health ofbreastfed infants. Strong evidences are available now to support theroles of HMOs on promoting the growth of beneficial gut bacteria;inhibiting the binding of pathogenic bacteria, human immunodeficiencyvirus (HIV), or protozoan parasites to gut epithelial cells; modulatingimmune responses; and influencing the functions of gut epithelium.

Among individual HMOs with known functions, disialyllacto-N-tetraose(DSLNT), but not its non-sialylated (LNT) or mono-sialylated(sialyllacto-N-tetraose b or LSTb, with a sialic acid α2-6-linked to aninternal glycan) analog, was previously identified as a specific humanmilk oligosaccharide (HMOs) component that is effective for preventingnecrotizing enterocolitis (NEC) in a neonatal rat model. Thehexasaccharide is presented at a level of 0.2-0.6 gram in a liter ofhuman milk. However, it is not presented in porcine milk, and either isnot presented or exists only in trace amount in bovine milk. Due to thelimited availability of human milk and the absence or the low abundanceof DSLNT in bovine milk, it is impractical to obtain the compound inlarge scale for potential clinical therapeutic applications. The presentinvention provides useful methods for preparing sialylated HMOs andnovel sialylated HMO-type oligosaccharides. The methods are based inpart on the surprising discovery that Photobacterium damselaeα2-6-sialyltransferase (Pd2,6ST) can be used to transfer sialic acidmoieties to internal monosaccharide subunits as well as to terminalmonosaccharide subunits in complex substrate sugars.

Accordingly, some embodiments of the invention provide a method ofpreparing a sialylated oligosaccharide having at least two sialic acidmoieties. The method includes forming a reaction mixture containing: asubstrate sugar; cytidine-5′-monophospho-sialic acid (CMP-sialic acid orCMP-Sia) or derivatives; and Photobacterium damselaeα2-6-sialyltransferase (Pd2,6ST) under conditions sufficient to form thesialylated oligosaccharide.

Any suitable substrate sugar can be used in the methods of theinvention. In some embodiments, the substrate sugar is a monosaccharide.In some embodiments, the substrate sugar is selected from adisaccharide, a trisaccharide, a tetrasaccharide, a pentasaccharide, ahexasaccharide, a heptasaccharide, an octasaccharide, a nonasaccharide,a decasaccharide, an undecasaccharide, a dodecasaccharide, atridecasaccharide, a tetradecasaccharide, a pentadecasaccharide, and ahexadecasaccharide.

The substrate sugar can contain a variety of monosaccharide subunits.The substrate sugar can contain, for example, one or more moietiesselected from substituted or unsubstituted glucose (Glc), substituted orunsubstituted glucose-1-phosphate (Glc-1-P), substituted orunsubstituted glucuronic acid (GlcA), substituted or unsubstitutedglucuronic acid-1-phosphate (GlcA-1-P), substituted or unsubstitutediduronic acid (IdoA), substituted or unsubstituted iduronicacid-1-phosphate (IdoA-1-P), substituted or unsubstitutedN-acetylglucosamine (GlcNAc), substituted or unsubstitutedN-acetylglucosamine-1-phosphate (GlcNAc-1-P), substituted orunsubstituted glucosamine (GlcNH₂), substituted or unsubstitutedglucosamine-1-phosphate (GlcNH₂-1-P), substituted or unsubstitutedgalactose (Gal), substituted or unsubstituted galactose-1-phosphate(Gal-1-P), substituted or unsubstituted galacturonic acid (GalA),substituted or unsubstituted galacturonic acid-1-phosphate (GalA-1-P),substituted or unsubstituted N-acetylgalactosamine (GalNAc), substitutedor unsubstituted N-acetylgalactosamine-1-phosphate (GalNAc-1-P),substituted or unsubstituted galactosamine (GalNH₂), substituted orunsubstituted galactosamine-1-phosphate (GalNH₂-1-P), substituted orunsubstituted mannose (Man), substituted or unsubstitutedmannose-1-phosphate (Man-1-P), and substituted or unsubstitutedN-acetylmannosamine (ManNAc), substituted or unsubstitutedN-acetylmannosamine-1-phosphate (ManNAc-1-P), substituted orunsubstituted mannosamine (ManNH₂), substituted or unsubstitutedmannosamine-1-phosphate (ManNH₂-1-P), and substituted or unsubstituted2-keto-3-deoxy-D-glycero-D-galactonononic acid (KDN).

In some embodiments, the substrate sugar contains one or more moietiesselected from substituted or unsubstituted galactose (Gal) andsubstituted or unsubstituted N-acetylgalactosamine (GalNAc).

In some embodiments, the substrate sugar is selected from galactose,lactose, N-acetyllactosamine, Galβ1-3GalNAc, Galβ1-3GlcNAc, Galα1-3Gal,Galα1-4Gal, Galα1-3Lac, Galα1-4Lac, Galβ1-4LacNAc,Galβ1-4GlcNAcβ1-3Galβ1-4Glc, Galβ1-3GlcNAcβ1-3Galβ1-4Glc,GalNAcβ1-4Galβ31-4Glc, Neu5Acα2-3(GalNAcβ1-4)Galβ1-4Glc,GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcA, Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc,GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Gal, and Galβ1-9KDN.

In some embodiments, the substrate sugar is prepared by any of themethods for preparing oligosaccharides described above.

Sialic acid is a general term for N- and O-substituted derivatives ofneuraminic acid, and includes, but is not limited to, 5-hydroxyl (Kdn),N-acetyl (Neu5Ac), or N-glycolyl (Neu5Gc) derivatives, as well asO-acetyl, O-lactyl, O-methyl, O-sulfate and O-phosphate derivatives. Insome embodiments, the sialic acid can be a compound of the formula:

wherein R¹ is selected from H, OH, N₃, NHC(O)Me, NHC(O)CH₂OH,NHC(O)CH₂N₃, NHC(O)OCH₂C═CH₂, NHC(O)OCHC═CH, NHC(O)CH₂F, NHC(O)CH₂NHCbz,NHC(O)CH₂OC(O)Me, and NHC(O)CH₂OBn; and R², R³, and R⁴ are independentlyselected from H, OH, N₃, OMe, F, OSO₃ ⁻, OPO₃H⁻, and OC(O)Me. In someembodiments, the CMP-sialic acid is cytidine 5′-monophosphateN-acetylneuraminic acid (CMP-Neu5Ac) or a CMP-Neu5Ac analog. Othersialic acid forms are useful in the methods of the present invention. Insome embodiments, the sialic acid can be a compound of the formula:

In some embodiments, the sialic acid can be a compound of the formula:

The CMP-sialic acid can be prepared prior to preparation of theoligosaccharide, or prepared in situ immediately prior to preparation ofthe oligosaccharide. In some embodiments, the method of the presentinvention also includes forming a reaction mixture including aCMP-sialic acid synthetase, cytidine 5′-triphosphate, andN-acetylneuraminic acid (Neu5Ac) or a Neu5Ac analog, under conditionssuitable to form CMP-Neu5Ac or a CMP-Neu5Ac analog. Any suitableCMP-sialic acid synthetase (i.e., N-acylneuraminatecytidylyltransferase, EC 2.7.7.43) can be used in the methods of theinvention. For example, CMP-sialic acid synthetases from E. coli, C.thermocellum, S. agalactiae, or N. meningitidis can be used. In someembodiments, the step of forming the CMP-sialic acid and the step offorming the sialylated oligosaccharide are performed in one pot.

In some embodiments, the sialic acid moiety of the CMP-sialic acid isprepared separately prior to use in the methods of the presentinvention. Alternatively, the sialic acid moiety can be prepared in situimmediately prior to use in the methods of the present invention. Insome embodiments, the method also includes forming a reaction mixtureincluding a sialic acid aldolase, pyruvic acid or derivatives thereof,and N-acetylmannosamine or derivatives thereof, under conditionssuitable to form Neu5Ac or a Neu5Ac analog. Any suitable sialic acidaldolase (i.e., N-Acetylneuraminate pyruvate lyase, EC 4.1.3.3) can beused in the methods of the invention. For example, sialic acid aldolasesfrom E. coli, L. plantarum, P. multocida, or N. meningitidis can beused. In some embodiments, the step of forming the sialic acid moiety,the step of forming the CMP-sialic acid, and the step of forming thesialylated oligosaccharide are performed in one pot.

In some embodiments, the CMP-sialic acid is prepared by a processincluding: i) forming a reaction mixture containing a CMP-sialic acidsynthetase, cytidine triphosphate, and sialic acid (Sia) underconditions sufficient to form the CMP-sialic acid.

In some embodiments, the sialic acid is prepared by a processingincluding: ii) forming a reaction mixture containing a sialic acidaldolase, pyruvic acid or a salt thereof, and N-acetylmannosamine orderivatives under conditions sufficient to form the sialic acid.

In some embodiments, steps i) and ii) are conducted in one pot.

In some embodiments, the sialylated oligosaccharide has a structureaccording to Formula 1:

wherein

-   -   each of A, B, C, and D is a monosaccharide moiety independently        selected from Gal, GalNAc, Glc, GlcNAc, GlcA, and KDN;    -   each R is a sialic acid moiety;    -   each of subscripts q, r, and s is independently selected from 0        and 1; and    -   each of subscripts u, v, w, x, y, and z is selected from 0 and        1, and the sum of subscripts u, v, w, x, y, and z is equal to 2.

In some embodiments, A is independently selected from Gal, GalNAc, andGlcNAc; B is independently selected from Gal, GalNAc, Glc, GlcNAc, GlcA,and KDN; C is independently selected from Gal, GalNAc, Glc, and GlcNAc;and D is independently selected from Gal, Glc, GlcNAc, and GlcA.

In some embodiments, A is Gal; and each of subscripts q, r, and s is 0.

In some embodiments, the moiety A-B is selected from Galβ1-4Glc,Galβ1-4GlcNAc, Galβ1-3GalNAc, Galβ1-3GlcNAc, Galα1-3Gal, Galα1-4Gal, andGalβ1-9KDN; subscript q is 1; and each of subscripts r and s is 0.

In some embodiments, the moiety A-B-C is selected fromGalα1-3Galβ1-4Glc, Galα1-4Galβ1-4Glc, Galβ1-4Galβ1-4GlcNAc,GalNAcβ1-4Galβ1-4Glc, and GalNAcβ1-4Galβ1-4Glc; each of subscripts q andr is 1; and subscript s is 0.

In some embodiments, the moiety A-B-C-D is selected fromGalβ1-4GlcNAcβ1-3Galβ1-4Glc, Galβ1-3GlcNAcβ1-3Galβ1-4Glc,GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcA, Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc, andGlcNAcβ1-3Galβ1-4GlcNAcβ1-3Gal; and each of subscripts q, r, and s is 1.

In some embodiments, each R is independently selected from an α2-3linked Neu5Ac moiety, an α2-6 linked Neu5Ac moiety, and an α2-8 linkedNeu5Ac moiety.

In some embodiments, the sialylated oligosaccharide is selected from:

-   -   Neu5Acα2-3(Neu5Acα2-6)Galβ1-4GlcNAc;    -   Neu5Acα2-6Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc;    -   Neu5Acα2-6Galβ1-4(Neu5Acα2-6)Galβ1-4GlcNAc;    -   Neu5Acα2-6Galβ1-3GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc;    -   Neu5Acα2-6Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4GlcNAc;    -   GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Gal;    -   Neu5Acα2-6GalNAcβ1-4GlcAcβ1-3(Neu5Acα2-6)GalNAcβ1-4GlcA;    -   Neu5Acα2-3(Neu5Acα2-6)Gal;    -   Neu5Acα2-3(Neu5Acα2-6)Galβ1-4Glc;    -   Neu5Acα2-8Neu5Acα2-3 Galβ1-4Glc;    -   Neu5Acα2-8Neu5Acα2-6 Galβ1-4Glc;    -   Neu5Acα2-3(Neu5Acα2-6)Galβ1-3GalNAc;    -   Neu5Acα2-6Galβ1-3(Neu5Acα2-6)GalNAc;    -   Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAc;    -   Neu5Acα2-3(Neu5Acα2-6)Galβ1-3GlcNAc;    -   Neu5Acα2-6Galα1-3(Neu5Acα2-6)Gal;    -   Neu5Acα2-6Galα1-4(Neu5Acα2-6)Gal;    -   Neu5Acα2-6Galα1-3 (Neu5Acα2-6)Galβ1-4Glc;    -   Neu5Acα2-6Galα1-4(Neu5Acα2-6)Galβ1-4Glc;    -   Neu5Acα2-6GalNAcβ1-4(Neu5Acα2-6)Galβ1-4Glc;    -   Neu5Acα2-3GalNAcβ1-4(Neu5Acα2-6)Galβ1-4Glc; and    -   Neu5Acα2-3(Neu5Acα2-6)Galβ1-9KDN.

The methods of the invention include forming reaction mixtures thatcontain Photobacterium damselae α2-6-sialyltransferase (Pd2,6ST). ThePd2,6ST can be, for example, purified prior to addition to the reactionmixture or secreted by a cell present in the reaction mixture.Alternatively, the Pd2,6ST can catalyze the reaction within a cellexpressing the enzyme.

Reaction mixtures can contain additional reagents for use inglycosylation techniques. For example, in certain embodiments, thereaction mixtures can contain buffers (e.g.,2-(N-morpholino)ethanesulfonic acid (MES),2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES),3-morpholinopropane-1-sulfonic acid (MOPS),2-amino-2-hydroxymethyl-propane-1,3-diol (TRIS), potassium phosphate,sodium phosphate, phosphate-buffered saline, sodium citrate, sodiumacetate, and sodium borate), cosolvents (e.g., dimethylsulfoxide,dimethylformamide, ethanol, methanol, tetrahydrofuran, acetone, andacetic acid), salts (e.g., NaCl, KCl, CaCl₂, and salts of Mn²⁺ andMg²⁺), chelators (e.g., ethyleneglycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA),2-({2-[Bis(carboxymethyl)amino]ethyl}(carboxymethyl)amino)acetic acid(EDTA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid(BAPTA)), reducing agents (e.g., dithiothreitol (DTT), β-mercaptoethanol(BME), and tris(2-carboxyethyl)phosphine (TCEP)), and labels (e.g.,fluorophores, radiolabels, and spin labels). Buffers, cosolvents, salts,chelators, reducing agents, and labels can be used at any suitableconcentration, which can be readily determined by one of skill in theart. In general, buffers, cosolvents, salts, chelators, reducing agents,and labels are included in reaction mixtures at concentrations rangingfrom about 1 μM to about 1 M. For example, a buffer, a cosolvent, asalt, a chelator, a reducing agent, or a label can be included in areaction mixture at a concentration of about 1 μM, or about 10 μM, orabout 100 μM, or about 1 mM, or about 10 mM, or about 25 mM, or about 50mM, or about 100 mM, or about 250 mM, or about 500 mM, or about 1 M.

Reactions are conducted under conditions sufficient to form thesialylated oligosaccharide. Generally, the conditions are sufficient totransfer the sialic acid from the CMP-sialic acid to the substratesugar. The reactions can be conducted at any suitable temperature. Ingeneral, the reactions are conducted at a temperature of from about 4°C. to about 40° C. The reactions can be conducted, for example, at about25° C. or about 37° C. The reactions can be conducted at any suitablepH. In general, the reactions are conducted at a pH of from about 5.5 toabout 10. The reactions can be conducted, for example, at a pH of fromabout 6.5 to about 9. The reactions can be conducted for any suitablelength of time. In general, the reaction mixtures are incubated undersuitable conditions for anywhere between about 1 minute and severalhours. The reactions can be conducted, for example, for about 1 minute,or about 5 minutes, or about 10 minutes, or about 30 minutes, or about 1hour, or about 2 hours, or about 4 hours, or about 8 hours, or about 12hours, or about 24 hours, or about 48 hours, or about 72 hours. Otherreaction conditions can be employed in the methods of the invention,depending on the identity of the particular CMP-sialic acid or substratesugar. Additional aspects of sialylation reactions are described in WO2013/022836, WO 2013/070677, and US 2013/0196385, the entirety of whichpublications are incorporated herein by reference in their entirety.

In a related aspect, the invention provides a sialylated oligosaccharideprepared according to any of the methods described herein. In certainembodiments, the invention provides novel sialylated saccharides. Insome embodiments, the sialylated oligosaccharide is selected from:

-   -   Neu5Acα2-3(Neu5Acα2-6)Galβ1-4Glc;    -   Neu5Acα2-8Neu5Acα2-3 Galβ1-4Glc;    -   Neu5Acα2-3(Neu5Acα2-6)Galβ1-3GalNAc;    -   Neu5Acα2-3(Neu5Acα2-6)Galβ1-3GalNAc;    -   Neu5Acα2-6Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc (DSLNnT);    -   Neu5Acα2-6Galβ1-3GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc; and    -   Neu5Acα2-3(Neu5Acα2-6)Galβ1-9KDN.

In some embodiments, the sialylated oligosaccharide is selected from:

-   -   Neu5Acα2-6Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc; and    -   Neu5Acα2-6Galβ1-3GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc

In some embodiments, the sialylated oligosaccharide isNeu5Acα2-6Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc.

VII. Methods for Treating Necrotizing Enterocolitis

In another aspect, the invention provides a method for treatingnecrotizing enterocolitis (NEC). The method includes administering to asubject in need thereof a sialylated oligosaccharide of the invention.

Necrotizing enterocolitis refers to a condition most often observed invery young infants, newborn infants, and prematurely-born infants.Possible causes include decreased blood flow to the bowel and reducedproduction of protective mucus, as well as bacteria in the intestine.NEC is considered to be the most serious gastrointestinal disorder amongpreterm infants.

The sialylated oligosaccharides can be administered at any suitable dosein the methods of the invention. In general, the sialylatedoligosaccharides are administered at a dose ranging from about 1microgram to about 1000 milligrams per kilogram of a subject's bodyweight (i.e., about 1 μg/kg-1000 mg/kg). The dose of a sialylatedoligosaccharide can be, for example, about 1 μg/kg-1 mg/kg, or about1-500 μg/kg, or about 25-250 μg/kg, or about 50-100 μg/kg. The dose of asialylated oligosaccharide can be about 0.1-1000 mg/kg, or about 1-500mg/kg, or about 25-250 mg/kg, or about 50-100 mg/kg. The dose of thesialylated oligosacharide can be about 1, 2, 3, 4, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 85, 90, 95, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 950μg/kg. The dose of the sialylated oligosacharide can be about 1, 2, 3,4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 85, 90,95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950 or 1000 mg/kg.

The dosages can be varied depending upon the requirements of thesubject, the severity of the disorder being treated, and the particularformulation being administered. The dose administered to a subjectshould be sufficient to result in a beneficial therapeutic response inthe subject. In certain instances, the dose administered to a subjectwill be sufficient to prevent the occurrence of NEC in the subject. Thesize of the dose will also be determined by the existence, nature, andextent of any adverse side-effects that accompany the administration ofthe compound in a particular subject. Determination of the proper dosagefor a particular situation is within the skill of the typicalpractitioner. Those of skill in the art are aware of the routineexperimentation that will produce an appropriate dosage range for asubject in need of treatment by oral administration or any other methodof administration of a compound, e.g., intravenous administration orparenteral administration, for example. Those of skill are also awarethat results provided by in vitro or in vivo experimental models can beused to extrapolate approximate dosages for a subject in need oftreatment. The total dosage can be divided and administered in portionsover a period of time suitable to treat NEC.

Administration of a sialylated oligosaccharide can be conducted for aperiod of time which will vary depending upon the nature of theparticular disorder, its severity and the overall condition of thesubject. Administration can be conducted, for example, hourly, every 2hours, three hours, four hours, six hours, eight hours, or twice dailyincluding every 12 hours, or any intervening interval thereof.Administration can be conducted once daily, or once every 36 hours or 48hours. Following treatment, a subject can be monitored for changes inhis or her condition and for alleviation of the symptoms of thedisorder. The dosage of the sialylated oligosaccharide can either beincreased in the event the subject does not respond significantly to aparticular dosage level, or the dose can be decreased if an alleviationof the symptoms of the disorder is observed, or if the disorder has beenablated, or if unacceptable side effects are seen with a particulardosage.

In a related aspect, the invention provides pharmaceutical compositionsfor the administration of the sialylated oligosaccharides. Thepharmaceutical compositions can be prepared by any of the methods wellknown in the art of pharmacy and drug delivery. In general, methods ofpreparing the compositions include the step of bringing the activeingredient into association with a carrier containing one or moreaccessory ingredients. The pharmaceutical compositions are typicallyprepared by uniformly and intimately bringing the active ingredient intoassociation with a liquid carrier or a finely divided solid carrier orboth, and then, if necessary, shaping the product into the desiredformulation. The compositions can be conveniently prepared and/orpackaged in unit dosage form.

Pharmaceutical compositions containing the sialylated oligosaccharidescan be in a form suitable for oral use. Suitable compositions for oraladministration include, but are not limited to, tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, syrups, elixirs, solutions, buccalpatches, oral gels, chewable tablets, and the like. Compositions fororal administration can be formulated according to any method known tothose of skill in the art. Such compositions can contain one or moreagents selected from sweetening agents, flavoring agents, coloringagents, antioxidants, and preserving agents in order to providepharmaceutically elegant and palatable preparations. The sialylatedoligosaccharides can be added, for example, to human milk, bovine milk,or infant formula.

Tablets generally contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients, including: inertdiluents, such as cellulose, silicon dioxide, aluminum oxide, calciumcarbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose,calcium phosphate, and sodium phosphate; granulating and disintegratingagents, such as corn starch and alginic acid; binding agents, such aspolyvinylpyrrolidone (PVP), cellulose, polyethylene glycol (PEG),starch, gelatin, and acacia; and lubricating agents such as magnesiumstearate, stearic acid, and talc. The tablets can be uncoated or coated,enterically or otherwise, by known techniques to delay disintegrationand absorption in the gastrointestinal tract and thereby provide asustained action over a longer period. For example, a time delaymaterial such as glyceryl monostearate or glyceryl distearate can beemployed. Tablets can also be coated with a semi-permeable membrane andoptional polymeric osmogents according to known techniques to formosmotic pump compositions for controlled release.

The pharmaceutical compositions can be in the form of a sterileinjectable aqueous or oleaginous solutions and suspensions. Sterileinjectable preparations can be formulated using non-toxicparenterally-acceptable vehicles including water, Ringer's solution, andisotonic sodium chloride solution, and acceptable solvents such as1,3-butane diol. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose any blandfixed oil can be employed including synthetic mono- or diglycerides. Inaddition, fatty acids such as oleic acid find use in the preparation ofinjectables.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients include, but are not limited to: suspending agents such assodium carboxymethylcellulose, methylcellulose,oleagino-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone,gum tragacanth and gum acacia; dispersing or wetting agents such aslecithin, polyoxyethylene stearate, and polyethylene sorbitanmonooleate; and preservatives such as ethyl, n-propyl, andp-hydroxybenzoate.

Oily suspensions can be formulated by suspending the active ingredientin a vegetable oil, for example, arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. These compositions can be preserved by theaddition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules (suitable for preparation of an aqueoussuspension by the addition of water) can contain the active ingredientin admixture with a dispersing agent, wetting agent, suspending agent,or combinations thereof. Additional excipients can also be present.

The pharmaceutical compositions of the invention can also be in the formof oil-in-water emulsions. The oily phase can be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, such as gum acacia or gum tragacanth;naturally-occurring phospholipids, such as soy lecithin; esters orpartial esters derived from fatty acids and hexitol anhydrides, such assorbitan monooleate; and condensation products of said partial esterswith ethylene oxide, such as polyoxyethylene sorbitan monooleate.

Compositions for oral administration can be formulated as hard gelatincapsules wherein the active ingredient is mixed with an inert soliddiluent (such as calcium carbonate, calcium phosphate, or kaolin), or assoft gelatin capsules wherein the active ingredient is mixed with wateror an oil medium (such as peanut oil, liquid paraffin, or olive oil).

Transdermal delivery of the sialylate oligosaccharides can beaccomplished by means of iontophoretic patches and the like. Thecompounds can also be administered in the form of suppositories forrectal administration. These compositions can be prepared by mixing thecompound with a suitable non-irritating excipient which is solid atordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum to release the compound. Such materialsinclude cocoa butter and polyethylene glycols.

VIII. EXAMPLES Example 1 Enzymes

NahK_ATCC15697 and NahK_ATCC55813 N-acetylhexosamine 1-Kinases

NahK (EC 2.7.1.162) catalyzes the direct addition of a phosphate fromadenosine 5′-triphosphate (ATP) to the anomeric position ofN-acetylhexosamine for the formation of N-acetylhexosamine-1-phosphateand adenosine 5′-diphosphate (ADP). The only characterized NahK to dateis encoded by the lnpB gene in the lnpABCD operon of Bifidobacteriumlongum JCM1217. Herein we report the cloning and characterization of twonew NahKs from Bifidobacterium infantis (ATCC15697) and Bifidobacteriumlongum (ATCC55813), respectively. A new capillary electrophoresis-basedassay method has been developed for biochemical characterization ofNahKs. We found that in addition to previously reported NahK substrates,various GlcNAc derivatives including those with C2-azido, C6-azido, and6-O-sulfate groups are tolerable substrates for the newly cloned NahKs.In addition, despite of their low activities toward glucose andgalactose, the activities of both NahKs are much higher for mannose andsome of its C-2, C-4, and C-6 derivatives including 2-deoxy-mannose or2-deoxy-glucose.

Experimental

Bacterial Strains, Plasmids, and Materials. Electrocompetent DH5α andchemically competent BL21 (DE3) E. coli cells were from Invitrogen(Carlsbad, Calif.). Bifidobacterium longum Reuter ATCC #55813 was fromAmerican Type Culture Collection (ATCC, Manassas, Va.). Genomic DNA ofBifidobacterium longum subsp. infantis (ATCC #15697) was a kind giftfrom Professor David Mills (University of California, Davis). Vectorplasmid pET22b(+) was from Novagen (EMD Biosciences Inc. Madison, Wis.).Ni²⁺-NTA agarose (nickel-nitrilotriacetic acid agarose), QIAprep spinminiprep kit, and QIAEX II gel extraction kit were from Qiagen(Valencia, Calif.). Herculase-enhanced DNA polymerase was fromStratagene (La Jolla, Calif.). T4 DNA ligase and 1 kb DNA ladder werefrom Promega (Madison, Wis.). NdeI and XhoI restriction enzymes werefrom New England Biolabs Inc. (Beverly, Mass.).Adenosine-5′-triphosphate disodium salt (ATP), GlcNAc, and GalNAc werefrom Sigma (St. Louis, Mo.). GlcNAc, GalNAc, mannose, and ManNAcderivatives were synthesized according to reported procedures.

Cloning. NahK_ATCC15697 and NahK_ATCC55813 were each cloned as aC-His₆-tagged (SEQ ID NO:22) fusion protein in pET22b(+) vector usinggenomic DNAs of Bifidobacterium longum subsp. infantis ATCC#15697 andBifidobacterium longumATCC#55813, respectively, as the template forpolymerase chain reactions (PCR). The primers used for NahK_ATCC15697were: forward primer 5′ ACCCCATATGAACAACACCAATGAAGCCCTG 3′ (SEQ IDNO:23) (Ndel restriction site is underlined) and reverse primer 5′TGACCTCGAGCTTGGTCGTCTCCATGACGTCG 3′ (SEQ ID NO:24) (XhoI restrictionsite is underlined). The primers used for NahK_ATCC55813 were: forwardprimer 5′ ACCCCATATGACCGAAAGCAATGAAGTTTTATTC 3′ (SEQ ID NO:25) (Ndelrestriction site is underlined) and reverse primer 5′TGACCTCGAGCCTGGCAGCCTCCATGATG 3′ (SEQ ID NO:26(XhoI restriction site isunderlined). PCR was performed in a 50 μL reaction mixture containinggenomic DNA (1 μg), forward and reverse primers (1 μM each),10×Herculase buffer (5 μL),dNTP mixture (1 mM), and 5 U (1 μL) ofHerculase-enhanced DNA polymerase. The reaction mixture was subjected to35 cycles of amplification with an annealing temperature of 52° C. Theresulting PCR product was purified and digested with Ndel and XhoIrestriction enzymes. The purified and digested PCR product was ligatedwith predigested pET22b(+) vector and transformed into electrocompetentE. coli DH5α cells. Selected clones were grown for minipreps andcharacterization by restriction mapping and DNA sequencing performed byDavis Sequencing Facility at the University of California-Davis.

Expression and purification. Positive plasmids were selected andtransformed into BL21(DE3) chemically competent cells. Theplasmid-bearing E. coli cells were cultured in LB rich medium (10 g/Ltryptone, 5 g/L yeast extract, and 10 g/L NaCl) supplied with ampicillin(100 μg/mL). Overexpression of the target protein was achieved byinducing the E. coli culture with 0.1 mM ofisopropyl-1-thio-β-D-galactopyranoside (IPTG) when the OD_(600 nm) ofthe culture reaches 0.8-1.0 followed by incubation at 20° C. for 24 hwith vigorous shaking at 250 rpm in a C25KC incubator shaker (NewBrunswick Scientific, Edison, NJ). To obtain the cell lysate, cells wereharvested by centrifuge cell culture at 4000×g for 2 h. The cell pelletwas re-suspended in lysis buffer (pH 8.0, 100 mM Tris-HCl containing0.1% Triton X-100, 20 mL/L cell culture) containing lysozyme (100 μg/mL)and DNaseI (3 μg/mL). After incubating at 37° C. for 60 min withvigorous shaking (250 rpm), the lysate was collected by centrifugationat 12,000 g for 30 min. His₆-tagged (SEQ ID NO:22) target proteins werepurified from cell lysate using an ÄKTA FPLC system (GE Healthcare,Piscataway, N.J., USA). To do this, the lysate was loaded to a HisTrap™FF 5 mL column (GE Healthcare) pre-washed and equilibrated with bindingbuffer (0.5 M NaCl, 20 mM Tris-HCl, pH 7.5). The column was then washedwith 8 volumes of binding buffer, 10 volumes of washing buffer (10 mMimidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.5) and eluted with 8 volumesof elute buffer (200 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.5).Fractions containing the purified enzyme were combined and dialyzedagainst dialysis buffer (Tris-HCl containing 10% glycerol, pH 7.5, 20mM) and stored at 4° C.

Quantification of Purified Protein. Protein concentration was determinedin a 96-well plate using a Bicinchoninic Acid (BCA) Protein Assay Kit(Pierce Biotechnology, Rockford, Ill.) with bovine serum albumin as aprotein standard. The absorbance of each sample was measured at 562 nmby a BioTek Synergy™ HT Multi-Mode Microplate Reader.

pH Profile by Capillary Electrophoresis (CE) Assays. Typical enzymaticassays were performed in a 20 μL reaction mixture containing a buffer(200 mM) with a pH in the range of 6.0-11.0, GlcNAc (1 mM), ATP (1 mM),MgCl₂ (5 mM), and a NahK (0.75 μM). Buffers used were: MES, pH 6.0;Tris-HCl, pH 7.0-9.0; CAPS, pH 10.0-11.0. Reactions were allowed toproceed for 10 min at 37° C. and were stopped by adding 20 μL of coldethanol to each reaction mixture. Samples were centrifuged and thesupernatants were analyzed by a P/ACE™ Capillary Electrophoresis (CE)system equipped with a Photodiode Array (PDA) detector (Beckman Coulter,Inc., Fullerton, Calif.). CE conditions were as follows: 75 μm i.d.capillary, 25 KV/80 μÅ, 5 s vacuum injections, monitored at 254 nm, therunning buffer used was sodium tetraborate (25 mM, pH 10.0).

Effect of MgCl₂ on the Enzymatic Activity. Different concentrations ofMgCl₂ were used in a Tris-HCl buffer (pH 8.0, 200 mM) containing GlcNAc(1 mM), ATP (1 mM), and a NahK (0.75 μM). Reactions were allowed toproceed for 10 min at 37° C. Reaction without MgCl₂ was used as acontrol.

Substrates Specificity Assays. GlcNAc, GalNAc, and their derivatives (1mM) were used as substrates in the presence of ATP (1 mM) and MgCl₂ (5mM) in a Tris-HCl buffer (pH 8.0, 200 mM) to analyze the substratespecificity of NahKs. Two concentrations (0.75 μM or 15 μM) of each NahKwere used and the reactions were allowed to proceed for 10 min (for 0.75μM NahK) or 30 min (for 15 μM NahK) at 37° C. For substrate specificitystudies of Glc, Gal, mannose, ManNAc, and their derivatives, 15 μM ofNahK was used for each reaction and the reactions were carried out at37° C. for 30 min. All other conditions were the same as for GlcNAc,GalNAc, and their derivatives.

Kinetics by CE Assays. Reactions were carried out in duplicate at 37° C.for 10 min in a total volume of 20 μL in Tris-HCl buffer (200 mM, pH7.5) containing MgCl₂ (1 mM), ATP, GlcNAc or GalNAc, and NahK (0.25 μMwhen GlcNAc and ATP were used as substrates, 0.5 μM when GalNAc and ATPwere used as substrates). Apparent kinetic parameters were obtained byvarying the ATP concentration from 0.1-5.0 mM (0.1 mM, 0.2 mM, 0.4 mM, 1mM, 2 mM, and 5 mM) at a fixed concentration of GlcNAc or GalNAc (1 mM),or varying the concentration of GlcNAc or GalNAc (0.1 mM, 0.2 mM, 0.4mM, 1 mM, 2 mM, and 5 mM) at a fixed concentration of ATP (1 mM) andfitting the data to the Michaelis-Menten equation using Grafit 5.0.

Results and Discussion

Cloning, expression, and purification. NahKs from BifidobacteriuminfantisATCC#15697 (NahK_ATCC15697) and Bifidobacterium longumATCC#55813(NahK_ATCC55813) were each cloned as a C-His₆-tagged (SEQ IDNO:22) fusion protein in a pET22b(+) vector. Sequence alignment (FIG. 1)indicates that NahK_ATCC55813 is almost identical to the NahK fromBifidobacterium longum JCM1217 (NahK_JCM1217, GenBank accession no.BAF73925) except for a single amino acid difference R348H (R is inNahK_JCM1217). In comparison, NahK_ATCC15697 shares 90% amino acidsequence identity with NahK_JCM1217.

Both NahKs were expressed by induction with 0.1 mM ofisopropyl-1-thio-β-D-galactopyranoside (IPTG) followed by incubation at20° C. for 24 h with vigorous shaking (250rpm). Up to 180 mg and 185 mgof Ni²⁺-column purified NahK_ATCC15697 and NahK_ATCC55813, respectively,could be obtained from one liter of E. coli culture. SDS-PAGE analysisshows that both purified proteins migrated to around 41 kDa, matchingwell to the calculated molecular weights of the translated His₆-tagged(SEQ ID NO:22) fusion proteins of 41.4 and 40.9 kDa for NahK_ATCC15697and NahK_ATCC55813, respectively.

Capillary Electrophoresis (CE) Assays. Based on the detection of ADP andATP in the reaction mixture by a UV detector, a capillaryelectrophoresis-based method was developed to directly measure theformation of ADP and N-acetylhexosamine-1-phosphate from ATP andN-acetylhexosamine for characterizing the activities of NahKs. Both ATPand ADP gave absorbance at 254 nm with equal signal responses.

pH Profile. As shown in FIG. 2, both NahKs are highly active in a pHrange of 7.0-8.0 with slight variations. The activities of both NahKsdrop quickly with either decrease of the pH to lower than 7.0 orincrease of the pH to higher than 8.0. About 50% of the optimal activitywas observed at pH 6.0 and pH 8.5 for NahK_ATCC15697. In comparison,about 70% of the optimal activity was observed at pH 6.0 and pH 8.5 forNahK_ATCC55813. The pH optima of these two enzymes are slight differentfrom that (pH 8.5) of NahK_JCM1217. Overall, the activity ofNahK_ATCC55813 is higher than that of NahK_ATCC15697 in the pH range of6.0-10.0 when GlcNAc was used as the substrate and the same molarconcentrations of the enzymes were used.

Effect of MgCl₂. Similar to NahK_JCM1217 and other kinases, bothNahK_ATCC15697 and NahK_ATCC55813 require a divalent metal ion foractivity. As shown in FIG. 3, the optimal concentration of Mg²⁺ wasdetermined to be 1 mM. The activities of both NahKs in the presence of0.5 mM of Mg²⁺ were about two thirds of those in the presence of 1.0 mMof Mg²⁺. Increasing the concentration of Mg²⁺ from 1 mM to 20 mM causeda slight decrease of the activities of both NahKs.

Kinetics. The apparent kinetic parameters shown in Table 1 indicate thatthe activities of two NahKs are close, with NahK_ATCC55813 having 16% or39% higher activity than NahK_ATCC15697 when GlcNAc or GalNAc was usedas the substrate in the presence of ATP. Overall, GlcNAc is a moreefficient (3.1-fold for NahK_ATCC15697 and 2.6-fold for NahK_ATCC55813)substrate than GalNAc for both NahKs due to relatively lower K_(m)values and higher (˜2-fold) k_(cat) values obtained when GlcNAc wasused. Using ATP and GlcNAc as the substrates, the K_(m) values of ATP(0.10±0.03 mM and 0.11±0.03 mM) and GlcNAc (0.06±0.01 mM) for both NahKsare lower than those for NahK_JCM1217 (0.172 mM for ATP and 0.118 mM forGlcNAc) determined by high performance ion chromatography (HPIC) with apulsed amperometric detector (DX500, Dionex Corporation, Sunnyvale,Calif.) using a Dionex CarboPac PA1 column (4 mm×250 mm). Thediscrepancies of the parameters may be due to the differences in theassay conditions used.

TABLE 1 Apparent kinetic parameters of NahKs. NahK1_ATCC15697NahK_ATCC55813 Enzymes k_(cat)/K_(m) Substrate K_(m) (mM) k_(cat) (s⁻¹)k_(cat)/K_(m) (s⁻¹ mM⁻¹) K_(m) (mM) K_(cat) (s⁻¹) (s⁻¹ mM⁻¹) ATP^(a)0.10 ± 0.03 1.1 ± 0.1 11.0 0.11 ± 0.03 1.3 ± 0.1 11.8 GlcNAc 0.06 ± 0.010.95 ± 0.01 15.8 0.06 ± 0.01 1.1 ± 0.1 18.3 ATP^(b) 0.08 ± 0.03 0.38 ±0.02  4.8 0.06 ± 0.02 0.48 ± 0.03  8.0 GalNAc 0.09 ± 0.05 0.46 ± 0.07 5.1 0.08 ± 0.03 0.57 ± 0.04  7.1 ^(a)The other substrate is GlcNAc;^(b)The other substrate is GalNAc.

Substrate Specificity. The substrate specificity studies using GlcNAc,GalNAc, and their derivatives (Table 2) indicate that both NahKs exhibitpromiscuous substrate specificity and have comparable levels of activitytoward GlcNAc and GalNAc derivatives. Compared to NahK_ATCC15697,NahK_ATCC55813 is more reactive towards non-modified GlcNAc (T2-1),GalNAc (T2-11), and some of their C2-modified derivatives with anN-trifluoroacetyl (GlcNTFA T2-2 and GalNTFA T2-12), an N-azidoacetylgroup (GlcNAcN₃ T2-3 and GalNAcN₃ T2-13), or an N-butanoyl group (GlcNBuT2-4 and GalNBu T2-14). Nevertheless, NahK_ATCC15697 is more reactivethan NahK_ATCC55813 for some of C2-modified GlcNAc and GalNAcderivatives such as those with a bulky N-benzoyl group (GlcNBz T2-5 andGalNBz T2-15) and a C2-azido group (GlcN₃ T2-6 and GalN₃ T2-16).NahK_ATCC15697 is also more reactive towards 2-amino-2-deoxy-glucose(GlcNH₂ T2-7), 2-N-sulfo-glucose (GlcNS T2-8), as well as C6-modifiedGlcNAc derivatives such as 6-deoxy-GlcNAc (GlcNAc6Me T2-9),6-azido-6-deoxy-GlcNAc (GlcNAc6N₃ T2-10), and 6-O-sulfo-GlcNAc (GlcNAc6ST2-17). Both C2 and C6-modified derivatives GlcNAc such as6-O-sulfo-N-trifluoroacetyl glucosamine (GlcNTFA6S T2-18) and6-O-sulfo-2-azido-2-deoxy glucose (GlcN₃ T2-19) as well as both C2 andC3-modified GlcNAc derivative 3-O-sulfo-2-azido-2-deoxy glucose (GlcN₃3ST2-20) are poor but acceptable substrates for both enzymes. Overall,some of the C2-modified GlcNAc and GalNAc (T2-1-T2-5 and T2-11-T2-14)are relatively good substrates for both NahKs with yields varied from5.2-42.3% in a 10 min reaction containing 0.75 μM of enzyme. Incomparison, other C2-modified GlcNAc and GalNAc (T2-6-T2-8, 15, T2-16),C6-(T2-9, T2-10, T2-17), both C2- and C6-(T2-18, T2-19), as well as bothC2- and C3-modified GlcNAc (T2-20) derivatives are poor but tolerablesubstrates for both NahKs and the assays have to be carried out for alonger reaction time (30 min) with a 20-fold higher concentration (15μM) of enzyme.

TABLE 2 Substrate specificity of NahKs using GlcNAc, GalNAc, and theirderivatives. Percentage Conversion (%) NahK ATCC156 NahK ATCC5581 97 3^(a)0.75 μM ^(b)15 μM ^(a)0.75 μM ^(b)15 μM

35.4 ± 0.1 NA 42.3 ± 0.2 NA

10.7 ± 0.9 NA 16.2 ± 0.9 NA

11.5 ± 1.0 NA 22.8 ± 0.4 NA

20.9 ± 0.6 NA 35.0 ± 2.0 NA

10.3 ± 0.4 NA  5.2 ± 0.2 NA

0 14.5 ± 0.1  0 7.0 ± 0.1

0 15.0 ± 0.1  0 8.4 ± 0.1

0 6.4 ± 0.2 0 4.0 ± 0.1

 4.4 ± 1.2 41.8 ± 0.3   2.1 ± 0.2 36.3 ± 0.3 

0 37.2 ± 0.5  0 23.4 ± 0.1  Percentage Conversion (%) NahK ATCC15 NahKATCC5581 697 3 ^(a)0.75 μM ^(b)15 μM ^(a)0.75 μM ^(b)15 μM

12.5 ± 0.1  NA 19.9 ± 0.1 NA

11.2 ± 1.6  NA 21.8 ± 0.2 NA

9.9 ± 0.6 NA 21.0 ± 1.2 NA

12.1 ± 0.3  NA 24.0 ± 0.1 NA

0 62.2 ± 1.0  0 51.9 ± 0.5 

0 7.6 ± 0.1 0 4.3 ± 0.1

0 11.7 ± 0.2  0 6.6 ± 0.1

0 7.2 ± 0.1 0 3.3 ± 0.2

0 6.9 ± 0.1 0 4.4 ± 0.1

0 4.9 ± 0.1 0 3.9 ± 0.1 NA: not assayed; ^(a)Reactions were allowed toproceed for 10 min at 37° C.; ^(b)Reactions were allowed to proceed for30 min at 37° C.

Among twenty compounds of GlcNAc, GalNAc and their derivatives tested,compounds T2-1, T2-3-T2-5, T2-9-T2-11, T2-13-T2-15 have been reportedbefore as suitable substrates for NahK_JCM1217 [11-12], while othercompounds including T2-2, T2-6-T2-8, T2-12, and T2-16-T2-20 are newlyidentified substrates for NahKs. It is worth to note that some of thesecompounds have negatively charged O-sulfate group at different positionsof GlcNAc or its derivatives.

Quite interestingly, the substrate specificity studies using glucose(Glc T3-21), galactose (Gal T3-28), mannose (Man T3-23),N-acetylmannosamine (ManNAc T3-29), and derivatives of mannose andManNAc (Table 3) indicate that while both Glc (T3-21) and Gal (T3-28)are poor substrates for both NahKs, 2-deoxy glucose (2-deoxyGlc T3-22)or 2-deoxymannose is a better substrate. In addition, mannose (T3-23),its 2-fluoro- (2F-Man T3-24) and 2-azido- (2N₃-Man T3-26) derivatives,as well as its 4-deoxy (4-deoxyMan T3-27) derivative are relatively goodsubstrates. In comparison, 2-methyl modification of mannose (2Me-ManT3-25) decreases its tolerance as the substrate for both NahKs. Quitesurprisingly, while ManNAc (T3-29) and some of its C-2 derivatives(T3-30-T3-32) are poor substrates for the NahKs,N-azidoacetylmannosamine (ManNAcN₃ T3-33, a C2-derivative of ManNAc) andits C6-derivative N-acetyl-6-O-methylmannosamine (ManNAc6OMe T3-34) arebetter substrates for both NahKs. Overall, except for 2-fluoro-mannose(2F-Man T3-24), NahK_ATCC15697 shows higher activity than NahK_ATCC55813for mannose, ManNAc, and their derivatives.

TABLE 3 Substrate specificity of NahKs using Glc, Gal, Man, ManNAc, andtheir derivatives. Percentage Conversion (%) Substrates NahK_ATCC15697NahK_ATCC55813

9.1 ± 0.1 4.7 ± 0.1

44.8 ± 0.2  28.4 ± 0.1 

68.0 ± 1.7  37.1 ± 0.4 

44.4 ± 0.2  47.0 ± 0.1 

9.4 ± 0.5 0

53.3 ± 0.1  40.2 ± 0.2 

37.1 ± 0.2  23.9 ± 0.1 

7.3 ± 0.2 4.4 ± 0.1

8.9 ± 0.1 5.5 ± 0.1

7.6 ± 0.1 5.4 ± 0.2

12.0 ± 0.1  9.1 ± 0.2

12.0 ± 0.4  7.4 ± 0.3

20.3 ± 0.3  18.6 ± 0.4 

32.6 ± 0.1  28.9 ± 0.1  The concentration of the enzyme used was 15 μM.Reactions were allowed to proceed at 37° C. for 30 min.AtGlcAK—Arabidopsis thaliana glucuronokinase (EC 2.7.1.43)Experimental

Cloning, expression, and purification. Full length Arabidopsisthalianaglucuronokinase (EC 2.7.1.43) (AtGlcAK) (encoded by gene GlcAK1,DNA GenBank accession number: GU599900; protein GenBank accessionnumber: NP_566144) was cloned in pET15b vector from a cDNA library ofArabidopsis thaliana and expressed as an N-His₆-tagged fusion protein.The primers used were: forward primer 5′ GGAATTCCATATGGATCCGAATTCCACGG3′ (SEQ ID NO:27) (NdeI restriction site is bold and underlined) andreverse primer 5′ CCGCTCGAGTCATAAGGTCTGAATGTCAGAATCATTC 3′ (SEQ IDNO:28) (XhoI restriction site is bold and underlined). The resulting PCRproducts were digested with restriction enzymes, purified, and ligatedwith pET15b vector predigested with Ndel and XhoI restriction enzymes.The ligated product was transformed into electrocompetent E. coli DH5αcells. Selected clones were grown for minipreps and positive clones wereverified by restriction mapping and DNA sequencing performed by DavisSequencing Facility. The DNA sequence of the insert matched to GlcAK1.

The plasmid was transformed into E. coli BL21 (DE3) chemically competentcells for protein expression. E. coli cells harboring the pET15b-AtGlcAKplasmid were cultured in LB medium (10 g/L tryptone, 5 g/L yeastextract, and 10 g/L NaCl) with ampicillin (100 μg/mL) at 37° C. withrigorous shaking at 250 rpm in a C25KC incubator shaker (New BrunswickScientific, Edison, N.J.) until the OD600 nm of the culture reached0.8-1.0. Overexpression of the targeted proteins was achieved by adding0.15 mM of isopropyl-1-thio-β-D-galactopyranoside (IPTG) followed byincubation at 18° C. for 20 h with rigorous shaking at 250 rpm.

His₆-tagged (SEQ ID NO:22) protein was purified from cell lysate usingNi²⁺-NTA affinity column. To obtain cell lysate, cells were harvested bycentrifugation at 4,000 rpm (Sorvall) at 4° C. for 2 h. The cell pelletwas resuspended in lysis buffer (pH 8.0, 100 mM Tris-HCl containing 0.1%Triton X-100). Lysozyme (100 μg/mL) and DNaseI (5μg/mL) were added tothe cell suspension. The mixture was incubated at 37° C. for 1 hr withvigorous shaking (200rpm). Cell lysate was obtained as the supernatantby centrifugation at 11,000 rpm (Sorvall) at 4° C. for 45 min.Purification was performed by loading the supernatant onto a Ni²⁺-NTAcolumn pre-equilibrated with 10 column volumes of binding buffer (10 mMimidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). The column was wash with10 column volumes of binding buffer and 10column volumes of washingbuffer (40 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). Protein ofinterest was eluted with Tris-HCl (pH 7.5, 50 mM) containing imidazole(200 mM) and NaCl (0.5 M). The fractions containing the purified enzymewere collected and dialyzed against Tris-HCl buffer (pH 7.5, 20 mM)containing 30% glycerol. Dialyzed proteins were stored at -20° C.Alternatively, fractions containing purified enzyme were dialyzedagainst Tris-HCl buffer (pH 7.5, 20 mM) and freeze dried. On average, 57mg of purified protein was obtained from 1 liter of cell culture.

Substrates Specificity Assays by Thin-Layer Chromatography (TLC).

Enzymatic assays were carried out in a total volume of 10 μL in Tris-HClbuffer (100 mM, pH 7.5) containing GlcA (or GalA, IdoA, xylose) (10 mM),ATP (20 mM), MgCl₂(20 mM), and AtGlcAK (22 μg). Reactions were allowedto proceed at 37° C. for 15 hr and monitored using thin-layerchromatographic (TLC) analysis using n-PrOH:H₂O:NH₄OH=7:4:2 (by volume)as a developing solvent. For visualizing compounds on TLC plate,p-anisaldehyde sugar stain was used.

LC-MS Assays for AtGlcAK Reactions. The AtGlcAK reaction mixtures abovewere also analyzed by LC-MS. 2 μL of sample was diluted 100 fold and 8μL was injected into a Waters spherisorb ODS-2 column (5 μm particles,250 mm length, 4.6 mm I.D.). The sample was eluted with 30% acetonitrilein H₂O with 0.1% formic acid and detected by ESI-MS in negative mode.

Results and Discussion

Substrates Specificity Assays. As shown in FIG. 25 and FIG. 26,thin-layer chromatography results and mass spectrometry (MS) resultsshowed that GlcA, GalA, and IdoA are acceptable substrates for AtGlcAKfor the formation of GlcA-1-P, GalA-1-P, and IdoA-1P, respectively.

PmGlmU—Pasteurella multocida Glucosaminyl Uridyltransferase

Glycosyltranferases are key enzymes for the formation ofoligosaccharides and glycoconjugates in nature. Mostglycosyltransferases require sugar nucleotides as donor substrates andcatalyze the transfer of monosaccharides from sugar nucleotides toacceptors in high regio- and stereoselective manner. Some carbohydratestructures contain post-glycosylational modifications (modifications oncarbohydrates and glycoconjugates which take place after the formationof glycosidic bonds). One strategy to obtain naturally existingoligosaccharides and glycoconjugates with modified sugar moieties is todevelop novel chemoenzymatic methods using structurally modifiedmonosaccharides as starting materials and carbohydrate biosyntheticenzymes (the simplest carbohydrate biosynthetic route usually involves amonosaccharide kinase, a nucleotidyltransferase, and aglycosyltransferase) with substrate promiscuities. Carbohydrates withnon-natural modifications can be synthesized similarly. Some of thesecompounds are potential drug candidates as they can effectivelyinterfere with carbohydrate-dependent biological processes.

Glycosaminoglycans including keratan sulfate, heparan sulfate, andheparin are N-acetylglucosamine (GlcNAc)-containing polysaccharides withpost-glycosylational modifications. While GlcNAc and 6-O-sulfo-GlcNAcare commonly found in kearatan sulfate, additional modified GlcNAc formssuch as N-sulfo- and 3-O-sulfo-GlcNAc are common for heparan sulfate andheparin. In addition, 6-O-sulfation on GlcNAc is also common in Lewis xand sialyl Lewis x structures and has been shown to affect the bindingaffinity of the related carbohydrate-binding proteins such as Selectinsand Siglecs. In attempts to synthesizing glycans containing naturallymodified GlcNAc and their non-natural derivatives usingglycosyltransferase-catalyzed reactions, we applied an efficient one-potthree-enzyme approach to synthesize UDP-GlcNAc derivatives includingUDP-6-O-sulfo-GlcNAc, UDP-GlcNTFA, and azido-containing UDP-GlcNAcderivatives. Additional UDP-GlcNAc derivatives, includingUDP-N-sulfo-glucosamine, were also produced by chemical diversificationfrom enzymatically produced UDP-GlcNAc derivatives. These compounds willbe tested as potential donor substrates for GlcNAc-glycosyltransferases.

Experimental

Cloning, expression, and purification of PmG1mU. The gene sequence ofPm1806 from Pasteurella multocida subsp. multocida strain Pm70 (GenBankaccession no. AAK03890) was used as a reference for designing primers.The genomic DNA of Pasteurella multocidastrain P-1059 (ATCC 15742) wasused as a template for polymerase chain reaction (PCR). Full lengthPasteurella multocida N-acetylglucosamine-1-phosphateuridylyltransferase (PmGlmU) was cloned in pET15b and pET22b(+) vectorsas N-His₆-(SEQ ID NO:22) and C-His₆-tagged (SEQ ID NO:22) fusionproteins, respectively. For cloning into pET15b vector as anN-His₆-tagged (SEQ ID NO:22) protein, the primers used were: forwardprimer 5′ GATCCATATG AAAGAGAAAGCATTAAGTATCGTG 3′ (SEQ ID NO:29) (Ndelrestriction site is bold and underlined) and reverse primer 5′CCGCTCGAGTTACTTTTTCGTTTGTTTAGTAGGGCG 3′ (SEQ ID NO:30) (XhoI restrictionsite is bold and underlined). For cloning into pET22b(+) vector as aC-His₆-tagged (SEQ ID NO:22) protein, the primers used were: forwardprimer 5′ GATCCATATGAAAGAGAAAGCATTAAGTATCGTG 3′ (SEQ ID NO:31) (Ndelrestriction site is bold and underlined) and reverse primer 5′ CCGCTCGAGCTTTTTCGTTTGTTTAGTAGGGCGTTGC 3′ (SEQ ID NO:32) (XhoI restriction site isbold and underlined). The resulting PCR products were digested withrestriction enzymes, purified, and ligated with pET15b or pET22b(+)vector predigested with NdeI and XhoI restriction enzymes. The ligatedproduct was transformed into electrocompetent E. coli DH5α cells.Selected clones were grown for minipreps and positive clones wereverified by restriction mapping and DNA sequencing performed by DavisSequencing Facility.

Positive plasmids were transformed into E. coli BL21 (DE3) chemicallycompetent cells. E. coli cells harboring the pET15b-PmGlmU orpET22b(+)-PmGlmU plasmid were cultured in LB medium (10 g/L tryptone, 5g/L yeast extract, and 10 g/L NaCl) with ampicillin (100 μg/mL) untilthe OD_(600 nm) of the culture reached 0.8-1.0. Overexpression of thetargeted proteins was achieved by adding 0.1 mM ofisopropyl-1-thio-β-D-galactopyranoside (IPTG) followed by incubation at25° C. for 18 h with rigorous shaking at 250 rpm in a C25KC incubatorshaker (New Brunswick Scientific, Edison, N.J.).

His₆-tagged (SEQ ID NO:22) proteins were purified from cell lysate usingNi²⁺-NTA affinity column. To obtain cell lysate, cells were harvested bycentrifugation at 4,000 rpm (Sorvall) at 4° C. for 2 hr. The cell pelletwas resuspended in lysis buffer (pH 8.0, 100 mM Tris-HCl containing 0.1%Triton X-100). Lysozyme (100 μg/mL) and DNaseI (5 μg/mL) were then addedto the cell suspension. The mixture was incubated at 37° C. for 1 hrwith vigorous shaking (200 rpm). Cell lysate was obtained as thesupernatant by centrifugation at 11,000 rpm (Sorvall) at 4° C. for 45min. Purification is performed by loading the supernatant onto aNi²⁺-NTA column pre-equilibrated with 10 column volumes of bindingbuffer (10 mM imidazole, 0.5 M NaCl, 50mM Tris-HCl, pH 7.5). The columnwas wash with 10 column volumes of binding buffer and 10 column volumesof washing buffer (40 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5).Protein of interest was eluted with Tris-HCl (pH 7.5,50 mM) containingimidazole (200 mM) and NaCl (0.5 M). The fractions containing thepurified enzymes were collected and dialyzed against Tris-HCl (pH 7.5,25mM) buffer containing 10% glycerol. Dialyzed proteins were stored at 4°C.

Results and Discussion

DNA and Protein Sequences of PmGlmU Cloned from Pasteurella multocidaStrain P-1059 (ATCC 15742). Compared to the sequences of GlmU (genePm1806) from Pasteurella multocida genomic strain Pm70 (GenBankaccession numbers: AE004439 for gene and AAK03890 for protein), thereare 13 base differences (C39A, T195C, A333G, G334A, T339C, G636A, G655A,G817C, T882A, A1006G, A1008T, G1071A, and G1266T) and four amino aciddifferences (E112K, D219N, E273Q, and T336A) (italicized and underlined)in Pasteurella multocida strain P-1059 (ATCC 15742).

Expression level and SDS-PAGE of PmGlmU. The N-His₆-tagged (SEQ IDNO:22) PmGlmU has a higher expression level than the C-His₆-tagged (SEQID NO:22) PmGlmU. On average, 170 mg of purified N-His₆-tagged (SEQ IDNO:22) PmG1mU was obtained from 1 liter of cell culture. SDS-PAGEanalysis shows that both the purified protein migrated to around 55 kDa.

BLUSP—Bifidobacterium longum UDP-sugar Pyrophosphorylase

Carbohydrates are widespread in nature and play pivotal roles inbiological systems. The key enzymes for the formation of glycosidicbonds in carbohydrates are glycosyltransferases. Mostglycosyltransferases require monosaccharide nucleotides as the commonactivated donor substrates. Among monosaccharide nucleotides used bymammalian glycosyltransferases, many are uridine 5′-diphosphate(UDP)-monosaccharides such as UDP-glucose (UDP-Glc), UDP-galactose(UDP-Gal), UDP-glucuronic acid (UDP-GlcA), UDP-N-acetylglucosamine(UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc), and UDP-xylose(UDP-Xyl). In addition, UDP-mannose (UDP-Man) has been isolated fromMycobacterium smegmatis and proposed to be an intermediate in thebiosynthesis of mycobacterial polysaccharides. Furthermore,UDP-N-acetylmannosamine (UDP-ManNAc) and UDP-N-acetylmannosaminuronicacid (UDP-ManNAcA) have been used by some bacteria for producingcapsular polysaccharides containing ManNAc or ManNAcA residues orforming ManNAcβ1-4GlcNAc-PP-undecaprenol (lipid II) for the biosynthesisof cell wall teichoic acids of Gram-positive bacteria.

The simplest biosynthetic route for obtaining monosaccharide nucleotidessuch as UDP-monosaccharides usually involves the formation of amonosaccharide-1-phosphate catalyzed by a monosaccharide-1-phosphatekinase followed by the formation of monosaccharide nucleotides catalyzedby a nucleotidyltransferase (or pyrophosphorylase). However, thesimplest route has not been applied routinely for the formation ofUDP-Gal due to the less common access to UTP:galactose-1-phosphateuridylyltransferases or UDP-Gal pyrophosphorylase (EC 2.7.7.10) fordirect formation of UDP-Gal from Gal-1-phosphate and UTP. For example,UDP-Gal used in galactosyltransferase-catalyzed enzymatic synthesis ofgalactosides has been more frequently obtained from UDP-Glc by reactionscatalyzed by UDP-Gal 4-epimerases or UDP-glucose:galactose-1-phosphateuridylyltransferases (EC 2.7.7.12, GalT or GalPUT) in the Leloirpathway.

Nevertheless, UDP-galactose pyrophosphorylase activity was identifiedfrom yeast Saccharomyces fragilis, pigeon liver, and mammalian livers.The enzyme was purified from bovine liver and Gram-positive bacteriumBifidobacterium bifidum. Recently, promiscuous UDP-sugarpyrophosphorylases (USPs) (EC 2.7.7.64) that can use variousmonosaccharide 1-phosphates in the presence of UTP for direct synthesisof UDP-monosaccharides including UDP-Glc, UDP-Gal, and UDP-GlcA, etc.were cloned from plants such as pea (Pisum sativum L.) sprouts (PsUSP)and Arabidopsis thaliana (AtUSP). Enzymes which share sequence homologyto plant USPs were also cloned from Leishmania major and Trypanosomacruzi, two trypanosomatid protozoan parasites, and were shown to havegood activity towards Gal-1-P and Glc-1-P and weaker activity towardsxylose-1-phosphate and GlcA-1-P. A USP with broad substrate specificityand optimal activity at 99° C. was also cloned from a hyperthermophilearchaea Pyrococcus furiosus DSM 3638 for which Glc-1-P, Man-1-P,Gal-1-P, Fuc-1-P, GlcNH₂-1-P, GalNH₂-1-P, and GlcNAc-1-P were all shownto be tolerable substrate, and both UTP and dTTP could be used asnucleotide triphosphate substrates by the enzyme. Nevertheless, none ofthese enzymes has been used in preparative-scale or large-scalesynthesis of sugar nucleotides and non-natural derivatives ofmonosaccharide-1-P have not been tested as substrates for DSPs.

Here we report the cloning of a promiscuous USP from a probioticBifidobacterium longum strain ATCC55813 and its application in anefficient one-pot three-enzyme system for preparative-scale synthesis ofUDP-monosaccharides and their derivatives from simple monosaccharides orderivatives (except for UDP-Glc which was synthesized from Glc-1-P in aone-pot two-enzyme system as discussed below). These compounds will betested as potential donor substrates for various glycosyltransferases.

Experimental

Cloning, expression, and purification of BLUSP. Full lengthBifidobacterium longum UDP-sugar pyrophosphorylase (EC 2.7.7.64) (BLUSP)(encoded by gene ugpA, DNA GenBank accession number: ACHI01000119, locustag: HMPREF0175_(—)1671; protein GenBank accession number: EEI80102) wascloned from the genomic DNA of Bifidobacterium longumstrain ATCC55813 inpET15b vector as an N-His₆-tagged (SEQ ID NO:22) fusion protein. Theprimers used were: forward primer 5′ GGAATTCCATATGACAGAAATAAACGATAAGGCC3′ (SEQ ID NO:33) (Ndel restriction site is bold and underlined) andreverse primer 5′ CGCGGATCCTCACACCCAATCGTCCG 3′ (SEQ ID NO:34) (BamHIrestriction site is bold and underlined). The resulting PCR productswere digested with restriction enzymes, purified, and ligated withpET15b vector predigested with Ndel and BamHI restriction enzymes. Theligated product was transformed into electrocompetent E. coli DH5αcells. Selected clones were grown for minipreps and positive clones wereverified by restriction mapping and DNA sequencing performed by DavisSequencing Facility. The DNA sequence of the insert matched to BL0739(ugpA) gene in the genomic sequence of Bifidobacterium longum NCC2705.Compared to the BL0739 (ugpA) gene sequence of Bifidobacterium longumNCC2705 (GenBank accession number: AE014295) which was annotated toencoding a hypothetical UTP:glucose-1-phosphate uridylyltransferase(GenBank accession number: AAN24556), there are 4 base differences(T35C, A47G, C228T, A465C) resulting in one amino acid difference (D16G)in the protein sequence of BLUSP.

The plasmid was transformed into E. coli BL21 (DE3) chemically competentcells for protein expression. E. coli cells harboring the pET15b-BLUSPplasmid were cultured in LB medium (10 g/L tryptone, 5 g/L yeastextract, and 10 g/L NaCl) with ampicillin (100 μg/mL) at 37° C. withrigorous shaking at 250 rpm in a C25KC incubator shaker (New BrunswickScientific, Edison, N.J.) until the OD_(600 nm) of the culture reached0.8-1.0. Overexpression of the targeted proteins was achieved by adding0.15 mM of isopropyl-1-thio-β-D-galactopyranoside (IPTG) followed byincubation at 18° C. for 20 hr with rigorous shaking at 250 rpm.

His₆-tagged (SEQ ID NO:22) protein was purified from cell lysate usingNi²⁺-NTA affinity column. To obtain cell lysate, cells were harvested bycentrifugation at 4,000 rpm (Sorvall) at 4° C. for 2 hr. The cell pelletwas resuspended in lysis buffer (pH 8.0,100 mM Tris-HCl containing 0.1%Triton X-100). Lysozyme (100 μg/mL) and DNaseI (5 μg/mL) were added tothe cell suspension. The mixture was incubated at 37° C. for 1 hr withvigorous shaking (200rpm). Cell lysate was obtained as the supernatantby centrifugation at 11,000 rpm (Sorvall) at 4° C. for 45 min.Purification was performed by loading the supernatant onto a Ni²⁺-NTAcolumn pre-equilibrated with 10 column volumes of binding buffer (10 mMimidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). The column was wash with10 column volumes of binding buffer and 10 column volumes of washingbuffer (40 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). Protein ofinterest was eluted with Tris-HCl (pH 7.5,50 mM) containing imidazole(200 mM) and NaCl (0.5 M). The fractions containing the purified enzymewere collected and dialyzed against Tris-HCl buffer (pH 7.5,25 mM)containing 10% glycerol and 0.25 M NaCl. Dialyzed proteins were storedat 4° C. Alternatively, fractions containing purified enzyme weredialyzed against Tris-HCl buffer (pH 7.5,25 mM) and freeze dried. Onaverage, 167 mg of purified protein was obtained from 1 liter of cellculture. Protein concentration was determined in a 96-well plate usingbicinchoninic acid with BSA as standard. The absorbance was measured at562 nm using a plate reader.

pH Profile Study for BLUSP. Typical enzymatic assays for pH profilestudies were carried out for 10 min at 37° C. in a total volume of 20 μLcontaining Glc-1-P (1 mM), UTP (1 mM), Mg²⁺ (20 mM), and BLUSP (10 ng)in a buffer (100 mM) with pH varying from 3.0 to 9.5. The reactionmixture was quenched by boiling for 5 min followed by adding 20 μL ofpre-chilled 95% (v/v) ethanol. The samples were then kept on ice untilanalyzed by a Beckman Coulter P/ACE MDQ Capillary Electrophoresis systemequipped with a UV detector and a 50 cm capillary tubing (75 μm I.D.,Beckman Coulter). Assays were run at 25 kV with 25 mM sodium boratebuffer (pH 9.8) for 22 min. Percent conversions were calculated frompeak areas of UDP-sugar and UTP monitored by UV absorbance at 254 nm.All assays were carried out in duplicate.

Effects of Metal Ions and EDTA. EDTA (5 mM), different concentrations(0.5, 1, 5, 10, 20, 50 mM) of MgCl₂, and various divalent metal cations(CaCl₂, CoCl₂, CuSO₄, MnCl₂, ZnCl₂) were used in a MES buffer (pH 6.5,100 mM) to analyze their effects on the uridylyltransferase activity ofBLUSP (10 ng in 20 μL total volume) using Glc-1-P (1 mM) as theacceptor. Other components are the same as those described for the pHprofile studies. Reaction without EDTA or metal ions was used as acontrol.

Capillary electrophoresis (CE) and thin-layer chromatograph (TLC) assaysfor kinase reactions. Kinase reactions were carried out at 37° C. in atotal volume of 30 μL in Tris-HCl buffer (100 mM, pH 8.0) containingmonosaccharide (15 mM), ATP (18 mM, 1.2 eq.), MgCl₂ (10 mM), and akinase (6 μg). These conditions were similar to those used forpreparative-scale synthesis. After 1 hr, 4 hr, and 24 hr, an aliquot of8 μL was withdrawn from each reaction mixture, boiled in a water bathfor 5 min and stored at −20° C. until being analyzed by capillaryelectrophoresis (CE) and TLC. For TLC analysis, 0.5 μL of each samplewas directly spotted on TLC plates, developed using suitable developingsolvents, and stained with anisaldehyde sugar stain. For CE analysis,1.5 μL of each sample was diluted into 30 μL and subjected to CEanalysis as described above for pH profile studies.

Results and Discussion

SDS-PAGE Analysis of BLUSP. SDS-PAGE analysis shows that the recombinantBLUSP has a very good expression level in E. coli and has a highsolubility. It consists of about 90% of the total protein extracts fromE. coli host cells and more than 90% of the soluble protein. The proteinsize observed is about 60 kDa which is close to 59.7 kDa calculatedmolecular weight.

pH Profile of BLUSP. As shown in FIG. 6, BLUSP is active in a broad pHrange of 4.0-8.0 and with optimal activity at pH 6.5 in MES buffer.

Effects of Metal Ions and EDTA. As shown in FIG. 7, a divalent metalcation such as Ca²⁺, Co²⁺, Mg²⁺, or Mn²⁺ is required for the activity ofBLUSP. BLUSP is inactive in the absence of a divalent metal cataion orin the presence of EDTA. At 20 mM concentration, Mg²⁺ was the best amongall divalent metal cations tested including Ca²⁺, Co²⁺, Mg²⁺, or Mn²⁺,Cu²⁺, and Zn²⁺. The optimal Mg²⁺ concentration for BLUSP activity wasfound to be 20 mM.

PmUgd—Pasteurella multocida UDP-Glucose Dehydrogenase

Cloning of PmUgd. PmUgd was cloned as a C-His₆-tagged (SEQ ID NO:22)fusion protein in pET22b(+) vector using the genomic DNA of P. multocidaP-1059 (ATCC#15742) as the template for polymerase chain reactions(PCR). Primers used for cloning were: forward primer5′-GATCCATATGAAGAAAATTACAATTGCTGGGGC-3′ (SEQ ID NO:35)(Ndel restrictionsite is underlined) and reverse primer 5′-CCGCTCGAGAGCATCACCGCCAAAAATATCTCTTG-3′(SEQ ID NO:36) (XhoI restrictionsite is underlined). PCR was performed in a reaction mixture of 50 μlcontaining genomic DNA (1 μg), forward and reverse primers (1 μM each),10×Herculase buffer (5 μl), dNTP mixture (1 mM), and 5 U (1 μl) ofHerculase-enhanced DNA polymerase. The reaction mixture was subjected to30 cycles of amplification with an annealing temperature of 55° C. Theresulting PCR products were purified, digested, and ligated with thecorresponding pre-digested vector. The ligation products weretransformed into electrocompetent E. coli DH5αcells. Plasmids containingthe target genes as confirmed by DNA sequencing (performed by UC-DavisSequencing Facility) were selected and transformed into E. coliBL21(DE3) chemically competent cells.

Compared to the DNA sequence of PM0776 gene from P. multocida strainPm70 (its genomic DNA sequence is available on NCBI), the obtained geneof PmUgd has 19 base differences (A357G, C381A, A390G, A397C, C404A,A406G, T408A, C414T, A420T, A426G, C430T, G438A, C447A, T451C, C453T,T456C, A464T, C582T, and G807A, the nucleotide before the number is fromthe DNA sequence of PM0776, the number is based on PM0776 gene) comparedto publically available PM0776 gene sequence. Furthermore, the C atposition 401 in PM0776 is missing in PmUgd and PmUgd has an extra Abetween 408 and 409 of PM0776. Overall, there are five amino aciddifferences in PmUgd (N127K, N133H, L1371, Y151H and Y155F, the aminoacid residue before the number is from the protein sequence deduced fromPM0776, the number is based on the protein sequence deduced from PM0776)compared to the deduced protein sequence from PM0776 gene.

Expression and Purification. E. coli strains were cultured in LB richmedium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl)supplemented with ampicillin (100 μg/mL). Over-expression of PmUgd wasachieved by inducing the E. coli BL21(DE3) cell culture with 0.1 mM ofisopropyl-1-thio-β-D-galactopyranoside (IPTG) when the OD_(600 nm) ofthe culture reached 0.8-1.0 followed by incubation at 20° C. for 20 h.

Bacterial cells were harvested by centrifugation at 4° C. in a SorvallLegend RT centrifuge with a hanging bucket rotor at 4000×rpm for 2 h.Harvested cells were resuspended in lysis buffer (Tris-HCl buffer, 100mM, pH 8.0 containing 0.1% Triton X-100) (20 mL for cells collected fromone liter cell culture). Lysozyme (100 μg/mL) and DNaseI (5 μg/mL) wereadded to the cell resuspension. The resulting mixture was incubated at37° C. for 1 h with shaking at 200 rpm. Cell lysate (supernatant) wasobtained by centrifugation at 12000×rpm for 15 min. Purification wascarried out by loading the supernatant onto a Ni²⁺-NTA columnpre-equilibrated with 8 column volumes of binding buffer (10 mMimidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). The column was washedwith 8 column volumes of binding buffer and 8 column volumes of washingbuffer (40 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). The targetprotein was eluted with Tris-HCl buffer (50 mM, pH 7.5) containingimidazole (200 mM) and NaCl (0.5 M). The fractions containing thepurified enzymes were collected and dialyzed against Tris-HCl buffer (20mM, pH 7.5) containing 10% glycerol. Dialyzed proteins were stored at 4°C.

About 23 mg PmUgd can be routinely expressed and purified from 1 L of E.coli culture under expression conditions described above.

PmHS1, PmHS2, KfiA

Materials and Methods

Bacterial Strains, Plasmids, and Materials. E. coli electrocompetentDH5α and chemically competent BL21 (DE3) cells were from Invitrogen(Carlsbad, Calif.). P. multocida P-934 (ATCC #12948) and P. multocidaP-1059 (ATCC #15742) were from American Type Culture Collection (ATCC,Manassas, Va., USA). KfiA synthetic gene with codons optimized for E.coli expression was synthesized by GeneArt (Grand Island, N.Y.) based onKfiA gene sequence from E. coli Nissle 1917 (GenBank accession number:AJ586888, ORF79). Vector plasmid pET15b was from Novagen (EMDBiosciences Inc. Madison, Wis., USA). Vector pMAL-c4X was purchased fromNew England Biolabs (Ipswich, Mass.). Nickel-nitrilotriacetic acidagarose (Ni²⁺-NTA agarose), QIAprep spin miniprep kit, and QIAEX II gelextraction kit were from Qiagen (Valencia, Calif., USA).Herculase-enhanced DNA polymerase was from Stratagene (La Jolla, Calif.,USA). T4 DNA ligase and 1 kb DNA ladder were from Promega (Madison,Wis., USA). NdeI, BamHI, EcoRI, and HindIII restriction enzymes werefrom New England Biolabs Inc. (Beverly, Mass., USA).

Cloning of PmHS1, PmHS2 and KfiA. PmHS2 was cloned as N- andC-His₆-tagged (SEQ ID NO:22) fusion proteins in pET15b and pET22b(+)vector, respectively, using genomic DNAs of P. multocida P-1059(ATCC#15742) as the template for polymerase chain reactions (PCR). PmHS1 and KfiA were cloned as a fusion protein of an N-terminal with amaltose-binding protein (MBP) and a C-terminal His₆ tag (SEQ ID NO:22)in pMAL-c4X vector using the P. multocida P-934 (ATCC#12948) and KfiAsynthetic gene as template, respectively. Primers used for cloning aresummarized in Table 8. PCR was performed in a reaction mixture of 50μLcontaining genomic DNA (1 μg), forward and reverse primers (1 μM each),10×Herculase buffer (5 μL), dNTP mixture (1 mM), and 5 U (1μL) ofHerculase-enhanced DNA polymerase. The reaction mixture was subjected to30 cycles of amplification with an annealing temperature of 55° C. (forPmHS1 and PmHS2) or 52° C. (for KfiA). The resulting PCR products werepurified, digested, and ligated with the corresponding pre-digestedvector. The ligation products were transformed into electrocompetent E.coli DH5α cells. Plasmids containing the target genes as confirmed byDNA sequencing (performed by UC-Davis Sequencing Facility) were selectedand transformed into E. coli BL21(DE3) chemically competent cells.

TABLE 4 Primers used for cloning PmHS1, PmHS2 and KfiA. PrimersSequences (5′-3′) (SEQ ID NO:) KfiA_pMAL-c4X_F_EcoRI GACC GAATTCATGATTGTTGCAAATATGAGC (37) KfiA_pMAL-c4X_R_HindIII GTCG AAGCTTTTAGTGGTGGTGGTGGTGGTGACCTT CCACATTATAC (38) PmHS1_pMAL-c4X _F_BamHI CGCGGATCC ATGAGCTTATTTAAACGTGCTAC (39) PmHS1_pMAL-c4X_R_HindIII GATC AAGCTT TTAGTGATGATGATGATGATGCTCGT TATAAAAAGATAAACACGG (40)PmHS2_pET15b/22b+_F_NdeI GATC CATATG AAGGGAAAAAAAGAGATGAC (41)PmHS2_pET15b_R_BamHI AAG GGATCC TTATAAAAAATAAAAAGGTAAACAGG (42)PmHS2_pET22b+_R_BamHI AAG GGATCC TTAGTGGTGGTGGTGGTGGTGTAAAAAATAAAAAGGTAAACAGG (43)

Expression and Purification. E. coli strains were cultured in LB richmedium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl)supplemented with ampicillin (100 μg/mL). Over-expression of PmHS1 andPmHS2 were achieved by inducing the E. coli BL21(DE3) cell culture with0.1 mM of isopropyl-1-thio-β-D-galactopyranoside (IPTG) when theOD_(600 nm) of the culture reached 0.8-1.0 followed by incubation at 20°C. for 20 h. Overexpression of KfiA was performed by inoculating 10 mLof a fresh overnight bacterial culture grown in LB containing 50 μg/mLampicillin and 20 μg/mL chloramphenicol into 1 L of LB (containing 50μg/mL of ampicillin, 20 μg/mL of chloramphenicol and 2 mg/mL ofL-arabinose). The culture was incubated at 37° C. with shaking at 250rpm. When the OD₆₀₀ of the culture reached 0.4-0.6, expression wasinduced by adding IPTG to a final concentration of 0.3 mM and then thecell was cultured at 20° C. for 20 h.

Bacterial cells were harvested by centrifugation at 4° C. in a SorvallLegend RT centrifuge with a hanging bucket rotor at 4000×rpm for 2 h.Harvested cells were resuspended in lysis buffer (Tris-HCl buffer, 100mM, pH 8.0 containing 0.1% Triton X-100) (20 mL for cells collected fromone liter cell culture). Lysozyme (100 μg/mL) and DNaseI (5 μg/in L)were added to the cell resuspension. The resulting mixture was incubatedat 37° C. for 1 h with shaking at 200 rpm. Cell lysate (supernatant) wasobtained by centrifugation at 12000×rpm for 15 min. Purification wascarried out by loading the supernatant onto a Ni²⁺-NTA columnpre-equilibrated with 10 column volumes of binding buffer (10 mMimidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5). The column was washedwith 10 column volumes of binding buffer and 10 column volumes ofwashing buffer (20-50 mM imidazole, 0.5 M NaCl, 50 mM Tris-HCl, pH 7.5).The target protein was eluted with Tris-HCl buffer (50 mM, pH 7.5)containing imidazole (200 mM) and NaCl (0.5 M). The fractions containingthe purified enzymes were collected and dialyzed against Tris-HCl buffer(20 mM, pH 7.5) containing 10% glycerol. Dialyzed proteins were storedat 4° C.

pH Profile by HPLC. Typical enzymatic assays were performed in a 10 μlreaction mixture containing a buffer (100 mM) with a pH in the range of4.0-10.0, UDP-GlcNAc (1 mM), GlcAβ132AA (1 mM), MnCl₂ (10 mM) and KfiA(9.0 μg) or PmHS2 (0.25 μg). Buffers used were: Na₂HPO₄/citric acid, pH4.0; MES, pH 5.0-6.5; TrisHCl, pH 7.0-9.0; and CAPS, pH 10.0. Reactionswere allowed to proceed for 30 min at 37° C. and were quenched by addingice-cold 10% (v/v) acetonitrile to make 100-fold dilutions. The sampleswere then kept on ice until an aliquot of 8 μl was injected and analyzedby a Shimadzu LC-2010A system equipped with a membrane on-line degasser,a temperature control unit and a fluorescence detector. A reverse phasePremier C18 column (250 9 4.6 mm I.D., 5 μm particle size, Shimadzu)protected with a C18 guard column cartridge was used. The mobile phasewas 25% (v/v) acetonitrile. The fluorescent compounds GlcAβ2AA andGlcNAcα1-4GlcAβ2AA were detected by excitation at 315 nm and emission at400 nm.

Effects of Metal Ions. Different concentrations (1, 5, 10, and 20 mM) ofMgCl₂ MnCl₂, CaCl₂, or CuCl₂ were used in a MES buffer (pH 6.5, 100 mM)to analyze their effects on the activity of KfiA (0.9 μg μl⁻¹) or PmHS2(2.5×10⁻² μg μl⁻¹). Reaction without metal ions was used as a control.The assay was performed as above pH profile.

Substrate Specificity of KfiA and PmHS2. All reactions were carried outin duplicate at 37° C. in MES (100 mM, pH 6.5) containing an UDP-GlcNAcor its derivatives (1 mM), GlcAα2AA (1 mM), MnCl₂(10 mM) and KfiA (1.08μg μl⁻¹) or PmHS2 (2.5×10⁻² μg μl⁻¹). At 30 min, 4 h or 16 h, aliquotsof reaction mixture were withdrawn and were quenched by adding ice-cold10% (v/v) acetonitrile to make 100-fold dilutions. The assays wereanalyzed by HPLC.

Results

Cloning, expression and purification of recombinant proteins. PmHS2 wascloned as an N- or a C-His₆-tagged (SEQ ID NO:22) protein using pET15band pET22b (+) vectors, respectively. Both N- and C-His₆-tagged (SEQ IDNO:22) proteins were able to be expressed as soluble forms in E. coliBL21(DE3) cells by induction 0.1 mM IPTG. Both could be easily purifiedusing Ni²⁺-affinity chromatography. The expression level of the solubleand active N-His₆-tagged (SEQ ID NO:22) form was relatively higher thanits C-His₆-tagged (SEQ ID NO:22) counterpart and N-His₆-PmHS2 wasstudied in detail. About 11 mg of N-His₆-PmHS2 was routinely obtainedfrom the cell lysate of one liter E. coli cell culture. KfiA wasexpressed in an N-terminal MBP and a C-terminal six-His fusion proteinin BL21(DE3) cells coexpressed with chaperone protein pGro7. Therecombinant KfiA was purified to homogeneity with a Ni²⁺-affinitycolumn. About 8.0 mg of MBP-KfiA-His₆ was routinely obtained from thecell lysate of one liter E. coli cell culture. Sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysisindicated that one-step Ni²⁺-column purification was efficient toprovide pure PmHS2 and KfiA. As expected from the calculated molecularweight of PmHS2 and KfiA, the size of the protein shown by SDS-PAGE wasabout 75 kDa and 69 kDa, respectively. To obtain a soluble and activerecombinant PmHS 1 in E. coli expression system, the MBP tag wasintroduced by using pMAL-c4X vector, while the C-His₆-tag (SEQ ID NO:22)was introduced by including the His₆-tag (SEQ ID NO:22) codons in the3′-primer used for cloning. Expression was achieved by incubating E.coli BL21(DE3) cells at 20° C. for 20 h with vigorous shaking (250 rpm)after the addition of IPTG (0.1 mM) for induction. Although it hasactivity, SDS-PAGE analysis indicated that only a small portion of therecombinant protein was seen in the cell lysate, the soluble portion ofthe cell extraction.

pH Profile of KfiA and the N-Acetylglucosaminyltransferase Activity ofPmHS2. As shown in FIG. 27, when GlcAB2AA was used as an acceptor, bothKfiA and PmSH2 were found to be active in a pH range of 5.0-9.0 with anoptimal activity at pH 5.0. The activities of both enzymes decreaseddramatically when pH was below 5.0.

Effects of Metal Ions on the Heparosan Synthase Activity of KfiA andPmHS2. The effects of different metal ions, Mg²⁺, Mn²⁺, Ca²⁺ and Cu²⁺ onthe heparosan synthase activity of KfiA and PmHS2 were investigated.Reaction without metal ions was used as a control. As shown in FIG. 28,no activity was detected without metals. Both enzymes showed bestactivities in the presence of Mn²⁺. Increasing Mn²⁺ from 1 mM to 20 mMincreased the activity of both KfiA and PmHS2 first and then decreasedthe activity of both enzymes. Compared with Mn²⁺, Mg²⁺ and Ca²⁺ showedmuch less efficiency for the activity of KfiA and PmHS2. No activity wasshown in the presence of Cu²⁺.

Substrate Specificity of KfiA and PmHS2. Using the HPLC method describedabove, the substrate specificities of KfiA and PmHS2 were examined usingGlcAa2AA and twenty two compounds of UDP-GlcNAc or UDP-Mannose or theirderivatives. Experimental data is shown in FIG. 11. Among the testedcompounds (see FIG. 12), both enzymes exhibited quite narrow substratespecificities. However, the catalytic efficiency of PmHS2 was much highthan that of KfiA. Both enzymes can use the UDP-GlcNAc (F12-3),UDP-GlcNTFA (F12-4), UDP-GlcNGc (F12-8), UDP-GlcNAcN₃ (F12-9), amongwhich the UDP-GlcNAc (F12-3) is the best substrate for both enzymes.Besides these four compounds, UDP-GlcNAc6N₃ (F12-5) is a substrate forPmHS2 but not for KfiA.

Example 2 Preparation of UDP-GlcNAc and Derivatives

General Methods for Compound Purification and Characterization.Chemicals were purchased and used without further purification. ¹H NMRand ¹³C NMR spectra were recorded on a 600 MHz NMR spectrometer. Highresolution electrospray ionization (ESI) mass spectra were obtained atthe Mass Spectrometry Facility in the University of California, Davis.Silica gel 60 Å (Sorbent Technologies) was used for flash columnchromatography. Analytical thin-layer chromatography (SorbentTechnologies) was performed on silica gel plates using anisaldehydesugar stain for detection. Gel filtration chromatography was performedwith a column (100 cm×2.5 cm) packed with BioGel P-2 Fine resins. ATP,UTP, and GlcNAc were purchased from Sigma. GlcNTFA, GlcN₃, GlcNAc6N₃,GlcNAc6S, GlcNS were synthesized as described previously. NanK_ATCC55813and PmPpA were overexpressed as discussed previously.

Synthesis of GlcNTFA6S T5b-6. GlcNTFA T5b-2 (300 mg, 1.09 mmol) wasdissolved in 15 mL of anhydrous DMF. Anhydrous Et₃N (5 mL) and sulfurtrioxide pyridine complex (1.2 eq.) were added at 0° C. After beingstirred at room temperature for overnight, the reaction was stopped byadding MeOH and concentrated. The residue was purified by flash columnchromatography (EtOAc:MeOH:H₂O=8:2:1, by volume) to afford6-O-sulfo-GlcNTFA T5b-6 (243 mg, 63%). ¹H NMR (600 MHz, D₂O) δ 5.25 (d,J=2.4 Hz, 0.6H), 4.84 (d, J=8.4 Hz, 0.4H), 4.26-3.51 (m, 6H). ¹³C NMR(150 MHz, D₂O) δ 159.75 (J=37.5 Hz), 159.69 (J=37.5 Hz), 117.01 (J=284.7Hz), 116.93 (J=284.7 Hz), 94.46, 80.59, 74.03, 73.24, 70.21, 70.00,69.84, 96.75, 67.22, 67.18, 57.34, 54.87.

Synthesis of GlcN₃6S T5b-7. 6-O-Sulfo-GlcN₃ T5b-7 was synthesized fromGlcN₃ T5b-3 (300 mg, 1.46 mmol) in 54% yield (224 mg) and the procedureswere similarly as described above for GlcNTFA6S T5b-6. ¹H NMR (600 MHz,D₂O) δ 5.35 (d, J=2.9 Hz, 0.4H), 4.71 (d, J=8.4 Hz, 0.6H), 4.21-3.82 (m,3H), 3.65-3.28 (m, 3H). ¹³C NMR (150 MHz, D₂O) δ 95.31, 91.39, 74.32,73.94, 71.55, 69.80, 69.61, 69.27, 67.15, 67.12, 66.85, 63.55.

One-Pot Three-Enzyme Synthesis of UDP-Sugars T5b-9-T5b-13. This wascarried out as shown in FIG. 4. Glucosamine derivatives T5b-1-T5b-5 (50to 300 mg, 1.0 eq.), ATP (1.2 eq.), and UTP (1.2 eq.) were dissolved inwater in a 50 mL centrifuge tube containing Tris-HCl buffer (100 mM, pH8.0) and MgCl₂ (10 mM). After the addition of appropriate amount ofNanK_ATCC55813 (3.2-4.8 mg), PmGlmU (5-7.5 mg), and PmPpA (2.5-5 mg),water was added to bring the volume of the reaction mixture to 20 mL.The reaction was carried out by incubating the solution in an isothermincubator for 24 hr to 48 hr at 37° C. with gentle shaking. Productformation was monitored by TLC (EtOAc:MeOH:H₂O=3:2:1 by volume) withp-anisaldehyde sugar staining. The reaction was stopped by adding thesame volume of ice-cold ethanol and incubating at 4° C. for 30 min. Themixture was concentrated and passed through a BioGel P-2 gel filtrationcolumn to obtain the desired product. Silica gel column purification(EtOAc:MeOH:H₂O=4:2:1) was applied when necessary to achieve furtherpurification.

One-pot three-enzyme synthesis of UDP-GlcNAc3N₃ was also conducted usingGlcNAc3N₃ as the staring sugar (See Table 5a) for reaction conditions).As shown in FIG. 29, the formation of UDP-GlcNAc3N₃ was confirmed byhigh-resolution mass spectrometry [HRMS (ESI) m/z calcd forC₁₇H₂₅N₆O₁₆P₂ (M−H) 631.0802, found 631.0817].

TABLE 5a Reaction conditions for the synthesis of UDP-GlcNAc3N₃.GlcNAc3N₃ (10 mM) Tris-HCl (pH 7.5) 100 mM MgCl₂ 10 mM ATP 20 mM UTP 20mM NahK_ATCC15697 17 μg PmGlmU 12.5 μg PmPpA 23 μg Total volume 20 μLreaction time/Temp 18 hr/37° C.

Uridine 5′-diphospho-2-acetamido-2-deoxy-α-D-glucopyranoside(UDP-GlcNAc, T5b-9). Yield, 81% (445 mg); white foam. ¹H NMR (600 MHz,D₂O) δ 7.97 (d, J=8.4 Hz, 1H), 5.97-6.00 (m, 2H), 5.53 (dd, J=6.6, 3.0Hz, 1H), 4.37-4.40 (m, 2H), 4.21-4.31 (m, 3H), 3.81-3.75 (m, 5H), 3.58(t, J=9.0 Hz, 1H), 2.09 (s, 3H). ¹³C NMR (150 MHz, D₂O) δ 174.94,166.39, 151.99, 141.82, 102.85, 94.68, 88.72, 83.40 (d, J=8.7 Hz),73.96, 73.20, 71.13, 69.83, 65.18, 65.15, 60.53, 53.88 (d, J=8.4 Hz),22.29. HRMS (ESI) m/z calcd for C₁₇H₂₇N₃O₁₇P₂ (M+H) 608.0894, found608.0906.

Uridine 5′-diphospho-2-deoxy-2-trifluoroacetamido-α-D-glucopyranoside(UDP-GlcNTFA, T5b-10). Yield, 97% (699 mg); white foam. ¹H NMR (600 MHz,D₂O) δ 7.95 (d, J=7.8 Hz, 1H), 5.97-5.98 (m, 2H), 5.64 (dd, J=6.6, 3.0Hz, 1H), 4.35-4.39 (m, 2H), 4.18-4.29 (m, 3H), 4.12 (d, J=10.8 Hz, 1H),3.93-3.98 (m, 2H), 3.91 (dd, J=12.6, 1.8 Hz, 1H), 3.85 (dd, J=12.0, 4.2Hz, 1H), 3.61 (t, J=9.0 Hz, 1H). ¹³C NMR (150 MHz, D₂O) δ 166.39, 159.73(d, J=37.5 Hz), 151.94, 141.83, 116.88 (d, J=284.6 Hz), 102.79, 93.91,88.79, 83.22 (d, J=9.0 Hz), 73.92, 73.23, 70.35, 69.76, 69.68, 65.12,60.42, 54.53 (d, J=8.9 Hz). HRMS (ESI) m/z calcd for C₁₇H₂₄F₃N₃O₁₇P₂(M+H) 662.0611, found 662.0615.

Uridine 5′-diphospho-2-azido-2-deoxy-α-D-glucopyranoside (UDP-GlcN₃,T5b-11). Yield, 54% (124 mg); white foam. ¹H NMR (600 MHz, D₂O) δ 7.96(d, J=8.4 Hz, 1H), 5.97-5.96 (m, 2H), 5.68 (dd, J=7.2, 3 Hz, 1H),4.34-4.37 (m, 2H), 4.18-4.27 (m, 3H), 3.89-3.93 (m, 2H), 3.85 (dd,J=12.6, 2.4 Hz, 1H), 3.79 (dd, J=12.0, 4.2 Hz, 1H), 3.54 (t, J=9.6 Hz,1H), 3.38 (d, J=10.2 Hz, 1H). ¹³C NMR (150 MHz, D₂O) δ 166.39, 151.96,141.84, 102.79, 94.60, 88.64, 83.34 (d, J=9 Hz), 73.91, 73.07, 70.85,69.77, 69.49, 65.07, 62.93 (d, J=8.6 Hz), 60.29. HRMS (ESI) m/z calcdfor C₁₅H₂₃N₅O₁₆P₂ (M+H) 633.0959, found 633.0960.

Uridine 5′-diphospho-2-acetamido-6-azido-2,6-dideoxy-α-D-glucopyranoside(UDP-GlcNAc6N₃, T5b-12). Yield, 72% (462 mg); white foam. ¹H NMR (600MHz, D₂O) δ 7.93 (d, J=7.8 Hz, 1H), 5.96-5.94 (m, 2H), 5.15 (s, 1H),4.32-4.36 (m, 2H), 4.17-4.24 (m, 3H), 4.00-4.04 (m, 2H), 3.79 (t, J=9.6Hz, 1H), 3.72 (dd, J=13.2, 2.4 Hz, 1H), 3.55-3.62 (m, 2H), 2.06 (s, 3H).¹³C NMR (150 MHz, D₂O) δ 174.86, 166.33, 151.86, 141.82, 102.76, 94.53,88.92, 83.15 (d, J=8.9 Hz), 73.93, 71.85, 70.28, 70.28, 69.68, 65.16,53.77 (d, J=7.4 Hz), 50.71, 22.22. HRMS (ESI) m/z calcd forC₁₇H₂₆N₆O₁₆P₂ (M+H) 592.0693, found 592.0698.

Uridine 5′-diphospho-2-acetamido-2-deoxy-6-O-sulfo-α-D-glucopyranoside(UDP-GlcNAc6S, T5b-13). Yield, 62% (70 mg); white foam. ¹H NMR (600 MHz,D₂O) δ 7.96 (d, J=7.8 Hz, 1H), 5.97-5.99 (m, 2H), 5.55 (dd, J=7.2, 3.0Hz, 1H), 4.35-4.38 (m, 3H), 4.26-4.30 (m, 3H), 4.18-4.22 (m, 1H), 4.12(d, J=9.6 Hz, 1H), 4.04 (d, J=10.8 Hz, 1H), 3.84 (t, J=9.6 Hz, 1H), 3.68(t, J=9.6 Hz, 1H), 2.09 (s, 3H). ¹³C NMR (150 MHz, D₂O) δ 174.84,166.40, 151.93, 141.73, 102.76, 94.57, 88.72, 83.15 (d, J=9.3 Hz),73.89, 70.83, 69.70, 69.04, 66.56, 65.16, 65.13, 53.67 (d, J=8.1 Hz),22.17. HRMS (ESI) m/z calcd for C₁₇H₂₇N₃O₂₀P₂S (M+H) 688.0462, found688.0471.

Chemical Derivatization of UDP-Sugars F5-2F5-8, and F5-10F5-15.

Uridine 5′-diphospho-2-amino-2-deoxy-α-D-glucopyranoside (UDP-GlcNH₂,F5-2). UDP-GlcNTFA F5-1 (150 mg, 0.22 mmol) was dissolved in 25 mL ofmethanol and 5 mL of H₂O. The pH of the solution was adjusted to 9.5 byadding K₂CO₃. After being vigorously stirred at r.t. for overnight, thereaction mixture was neutralized with DOWEX HCR-W2 (H⁺) resin, filteredand concentrated. The residue was purified by flash columnchromatography (EtOAc:MeOH:H₂O=1:1:1, by volume) to afford UDP-GlcNH₂F5-2 as white solid in 98% yield (122 mg). ¹H NMR (600 MHz, D₂O) δ 7.90(d, J=7.8 Hz, 1H), 5.89-5.92 (m, 2H), 5.79 (dd, J=6.0, 3.0 Hz, 1H),4.30-4.32 (m, 2H), 4.16-4.24 (m, 3H), 3.86-3.90 (m 2H), 3.81 (dd,J=12.6, 1.8 Hz, 1H), 3.77 (dd, J=12.6, 4.2 Hz, 1H), 3.52 (t, J=9.6 Hz,1H), 3.33 (d, J=10.8 Hz, 1H). ¹³C NMR (150 MHz, D₂O) δ 166.40, 151.93,141.75, 102.71, 92.87, 88.74, 83.21 (d, J=9 Hz), 73.91, 73.39, 69.85,69.69, 69.16, 65.23, 60.09, 54.27 (d, J=8.4 Hz). HRMS (ESI) m/z calcdfor C₁₅H₂₅N₃O₁₆P₂ (M+H) 566.0788, found 566.0791.

Uridine 5′-diphospho-2-sulfoamino-2-deoxy-α-D-glucopyranoside(UDP-GlcNS, F5-3). UDP-GlcNH₂ F5-2 (50 mg, 0.082 mmol) was dissolved in30 mL of water. The pH of the solution was adjusted to 9.5 by adding 2 NNaOH (aq). Sulfur trioxide-pyridine complex (65 mg, 0.41 mmol) was addedin three equal portions during 35 minutes intervals at room temperature,and the pH was maintained at 9.5 throughout the whole process using 2 NNaOH (aq). After being stirred at r.t. for overnight, the reactionmixture was neutralized with DOWEX HCR-W2 (H⁺) resin, filtered,concentrated, and purified using silica gel column(EtOAc:MeOH:H₂O=3:2:1, by volume) to obtain the UDP-GlcNS F5-3 in 86%yield (46 mg). ¹H NMR (600 MHz, D₂O) δ 7.90 (d, J=7.8 Hz, 1H), 5.92-5.93(m, 2H), 5.71 (s, 1H), 4.31-4.33 (m, 2H), 4.16-4.23 (m, 3H), 3.73-3.86(m, 3H), 3.66 (t, J=9.6 Hz, 1H), 3.51 (t, J=9.6 Hz, 1H), 3.24 (d, J=9.6Hz, 1H). ¹³C NMR (150 MHz, D₂O) δ 166.49, 152.13, 141.99, 103.04, 95.50,88.86, 83.53 (J=8.9 Hz), 74.02, 73.06, 71.73, 69.98, 69.96, 65.38,60.73, 58.11 (J=9.2 Hz). HRMS (ESI) m/z calcd for C₁₅H₂₅N₃O₁₉P₂S (M+H)646.0356, found 646.0373.

Uridine 5′-diphospho-2-hydroxyacetamido-2-deoxy-α-D-glucopyranoside(UDP-GlcNGc, F5-5). To a solution of UDP-GlcNH₂ F5-2 (30 mg, 0.049 mmol)in CH₃CN—H₂O (30 mL, 1:1, v/v) in the presence of NaHCO₃ (40 mg, 0.49mmol), the Acetoxyacetyl chloride (6.9 μL, 0.098 mmol) in CH₃CN (5 mL)was added. The reaction mixture was stirred for 4 hours at 0° C. and wasneutralized with DOWEX HCR-W2 (Fr) resin, filtered, and concentrated.The residue was purified by flash column chromatography(EtOAc:MeOH:H₂O=5:2:1, by volume) to afford UDP-GlcNGcAc F5-4 in 95%yield (31 mg). ¹H NMR (600 MHz, D₂O) δ 7.99 (d, J=7.8 Hz, 1H), 6.03-6.04(m, 2H), 5.62 (dd, J=6.6, 3.6 Hz, 1H), 4.41-4.45 (m, 2H), 4.24-4.35 (m,3H), 4.13 (d, J=10.2 Hz, 1H), 4.01 (d, J=7.8 Hz, 1H), 3.86-3.96 (m, 3H),3.63 (t, J=9.6 Hz, 1H), 2.25 (s, 3H). UDP-GlcNGcAc F5-4 was dissolved indry methanol (50 mL) containing analytic amount of sodium methoxide. Theresulted mixture was stirred at r.t. for overnight. The reaction mixturewas then neutralized with DOWEX HCR-W2 (H⁺) resin, filtered, andconcentration to give product UDP-GlcNGc F5-5 in 98% yield (28 mg). ¹HNMR (600 MHz, D₂O) δ 7.92 (d, J=7.8 Hz, 1H), 5.93-5.95 (m, 2H), 5.52(dd, J=7.2, 3.0 Hz, 1H), 4.31-4.35 (m, 2H), 4.09-4.25 (m, 5H), 4.02 (d,J=10.2 Hz, 1H), 3.83-3.91 (m, 3H), 3.79 (dd, J=12.6, 4.2 Hz, 1H), 3.55(t, J=9.6 Hz, 1H). ¹³C NMR (150 MHz, D₂O) δ 175.47, 166.37, 151.92,141.74, 101.73, 94.36, 88.53, 83.28 (d, J=8.4 Hz), 73.86, 73.09, 70.69,69.71, 69.54, 65.05, 61.11, 60.36, 53.46 (d, J=7.7 Hz). HRMS (ESI) m/zcalcd for C₁₇H₂₇N₃O₁₈P₂ (M+H) 624.0843, found 624.0847.

Uridine 5′-diphospho-2-azidoxyacetamido-2-deoxy-α-D-glucopyranoside(UDP-GlcNAz, F5-6). Sodium azide (62 mg, 0.98 mmol) was dissolved in 5mL of distilled H₂O and the mixture was cooled to 0° C. Bromoacetic acid(68 mg, 0.49 mmol) was then added over 10 min and the reaction wasallowed to slowly warm up to r.t. for overnight. The reaction wasacidified to pH 1.0 and extracted three times with 5 mL of diethylether. The organic portions were combined, dried over MgSO₄ andconcentrated. The crude mixture was dissolved in 10 mL of CH₂Cl₂ and twodrops of DMF and cooled to 0° C. Oxalyl chloride (54 μL, 0.64 mmol) wasslowly added over 15 min using a syringe. The reaction was allowed towarm up to r.t. for overnight. The solvent was removed under reducedpressure to afford the crude oil azidoacetyl chloride. To a solution ofUDP-GlcNH₂ F5-2 (30 mg, 0.049 mmol) in CH₃CN—H₂O (30 mL, 1:1, v/v) inthe presence of NaHCO₃ (40 mg, 0.49 mmol), the azidoacetyl chloride inCH₃CN (5 mL) was added. The reaction mixture was stirred for 4 hours at0° C. and was neutralized with DOWEX HCR-W2 (H⁺) resin, filtered, andconcentrated. The residue was purified by flash column chromatography(EtOAc:MeOH:H₂O=5:2:1, by volume) to afford UDP-GlcNAz F5-6 in 68% yield(22 mg). ¹H NMR (600 MHz, D₂O) δ 7.92 (d, J=8.4 Hz, 1H), 5.91-5.94 (m,2H), 5.49 (dd, J=7.2, 3.6 Hz, 1H), 4.30-4.36 (m, 2H), 4.00-4.24 (m, 6H),3.75-3.89 (m, 4H), 3.53 (t, J=9.6 Hz, 1H). ¹³C NMR (150 MHz, D₂O) δ171.13, 166.41, 151.98, 141.86, 102.84, 94.59, 88.80, 83.34 (d, J=9.0Hz), 73.94, 73.25, 71.02, 69.81, 69.66, 65.24, 60.51, 53.94 (d, J=9.0Hz), 52.69, 51.80. HRMS (ESI) m/z calcd for C₁₇H₂₆N₆O₁₇P₂(M+H) 649.0908,found 649.0917.

Uridine 5′-diphospho-2-phenylacetamido-2-deoxy-α-D-glucopyranoside(UDP-GlcNAcPh, F5-7). 2-Phenylacetyl acid (33 mg, 0.25 mmol) wasdissolved in 10 mL of CH₂Cl₂ and two drops of DMF. The mixture wascooled to 0° C. Oxalyl chloride (28 μL, 0.33 mmol) was slowly added over15 min using a syringe. The reaction was allowed to warm up to r.t. forovernight. The solvent was then removed under reduced pressure to afford2-phenylacetyl chloride as a light pink solid. To a solution ofUDP-GlcNH₂ F5-2 (30 mg, 0.049 mmol) in CH₃CN—H₂O (30 mL, 1:1, v/v) inthe presence of NaHCO₃ (40 mg, 0.49 mmol), the 2-phenylacetyl chloridein CH₃CN (5 mL) was added. The reaction mixture was stirred for 4 hoursat 0° C. and was neutralized with DOWEX HCR-W2 (H⁺) resin, filtered, andconcentrated. The residue was purified by flash column chromatography(EtOAc:MeOH:H₂O=6:2:1, by volume) to afford white solid UDP-GlcNAcPhF5-7 in 79% yield (26 mg). ¹H NMR (600 MHz, D₂O) δ 7.83 (d, J=8.4 Hz,1H), 7.32-7.35 (m, 2H), 7.26-7.29 (m, 3H), 5.90 (d, J=3.6 Hz, 1H), 5.83(d, J=7.8 Hz, 1H), 5.54 (dd, J=6.6, 3.0 Hz, 1H), 4.16-4.30 (m, 5H),3.77-4.00 (m, 5H), 3.67 (s, 2H), 3.54 (t, J=9.6 Hz, 1H). ¹³C NMR (150MHz, D₂O) δ 175.33, 166.16, 151.75, 141.64, 135.13, 129.36, 128.90,127.26, 102.74, 94.77, 88.80, 83.08 (d, J=9 Hz), 73.80, 73.25, 70.97,69.74, 69.65, 65.07, 60.50, 53.89 (d, J=8.7 Hz), 42.21. HRMS (ESI) m/zcalcd for C₂₃H₃₁N₃O₁₇P₂ (M+H) 684.1207, found 684.1215.

Uridine5′-diphospho-2-(1,1′-biphenyl-4-yl)acetamido-2-deoxy-α-D-glucopyranoside(UDP-GlcNAcPh₂, F5-8). UDP-GlcNAcPh₂F5-8 was synthesized from UDP-GlcNH₂F5-2 using a similar procedure as described above for UDP-GlcNAcPh F5-7g except that the reagent 2-phenylacetyl acid was replaced by2-([1,1′-biphenyl]-4-yl)acetic acid. UDP-GlcNAcPh₂ F5-8 was obtained asa white solid in 82% yield (31 mg). ¹H NMR (600 MHz, D₂O) δ 7.69 (d,J=8.4 Hz, 1H), 7.64 (d, J=8.4 Hz, 2H), 7.59 (d, J=7.2 Hz, 2H), 7.45-7.47(m, 2H), 7.34-7.41 (m, 3H), 5.79 (d, J=4.2 Hz, 1H), 5.64 (d, J=7.2 Hz,1H), 5.53 (dd, J=6.6, 3.0 Hz, 1H) 4.14-4.19 (m, 5H), 4.01 (d, J=10.2 Hz,1H), 3.92 (d, J=9.6 Hz, 1H), 3.65-3.85 (m, 5H), 3.53 (t, J=9.0 Hz, 1H).¹³C NMR (150 MHz, D₂O) δ 175.19, 165.92, 151.44, 141.31, 140.08, 139.12,134.51, 129.90, 129.16, 127.75, 127.14, 126.80, 102.49, 94.76, 88.77,82.87 (d, J=8.6 Hz), 73.82, 73.22, 71.01, 69.70, 69.44, 64.89, 60.46,53.88 (d, J=8.4 Hz), 41.80. HRMS (ESI) m/z calcd for C₂₉H₃₅N₃O₁₇P₂(M+H)760.1520, found 760.1534.

Uridine 5′-diphospho-2-acetamido-6-amino-2,6-dideoxy-α-D-glucopyranoside(UDP-GlcNAc6NH₂, F5-10). UDP-GlcNAc6N₃ (T5b-12 or F5-9) (100 mg, 0.16mmol) was dissolved in MeOH—H₂O (10 mL, 1:1, v/v) and 20 mg of Pd/C wasadded. The mixture was shaken under H₂ gas (4 Bar) for 1 hr, filtered,and concentrated. The residue was purified by flash columnchromatography (EtOAc:MeOH:H₂O=3:2:1, by volume) to affordUDP-GlcNAc6NH₂ F5-10 in 96% yield (93 mg). ¹H NMR (600 MHz, D₂O) δ 7.90(d, J=8.4 Hz, 1H), 5.89-5.93 (m, 2H), 5.48 (dd, J=6.6, 3.0 Hz, 1H),4.30-4.32 (m, 2H), 4.20-4.23 (m, 2H), 4.08-4.15 (m, 2H), 3.99 (d, J=10.8Hz, 1H), 3.77 (t, J=9.6 Hz, 1H), 3.45 (d, J=13.2 Hz, 1H), 3.40 (t, J=9.6Hz, 1H), 3.11 (t, J=12.6 Hz, 1H), 2.02 (s, 3H). ¹³C NMR (150 MHz, D₂O) δ174.94, 166.39, 151.96, 141.84, 102.79, 94.33, 88.82, 83.29 (J=9.0 Hz),73.92, 71.82, 69.80, 69.28, 65.30, 53.64 (J=8.9 Hz), 40.70, 22.21. HRMS(ESI) m/z calcd for C₁₇H₂₈N₄O₁₆P₂ (M+H)⁻ 607.1054, found 607.1068.

Uridine5′-diphospho-2-acetamido-6-hydroxyacetamido-2,6-dideoxy-α-D-glucopyranoside(UDP-GlcNAc6NGc, F5-12). UDP-GlcNAc6NGcAc F5-11 was synthesized fromUDP-GlcNAc6NH₂ F5-10 using the same process as described above forUDP-GlcNAcNGcAc F5-4. UDP-GlcNAc6NGcAc F5-11 was obtained as a whitesolid in 91% yield (31 mg). ¹H NMR (600 MHz, D₂O) δ 7.91 (d, J=7.8 Hz,1H), 5.91-5.93 (m, 2H), 5.46 (dd, J=6.6, 3.0 Hz, 1H), 4.62 (s, 2H),4.30-4.34 (m, 2H), 4.14-4.24 (m, 3H), 3.95 (m, 2H), 3.76 (t, J=9.0 Hz,1H), 3.61 (dd, J=14.4, 6.0 Hz, 1H), 3.54 (dd, J=14.4, 2.4 Hz, 1H), 3.35(dd, J=14.4, 4.2 Hz, 1H), 2.15 (s, 3H), 2.03 (s, 3H). ¹³C NMR (150 MHz,D₂O) δ 174.85, 173.43, 170.60, 166.44, 151.95, 141.74, 102.70, 94.40,88.70, 83.16, 73.87, 71.33, 70.89, 70.68, 69.66, 65.05, 62.88, 53.67,39.59, 22.14, 20.13. UDP-GlcNAc6NGc F5-12 was synthesized fromUDP-GlcNAc6NGcAc F5-11 using the same process as described above forUDP-GlcNGc F5-5 and obtained as a white solid in 98% yield (29 mg). ¹HNMR (600 MHz, D₂O) δ 8.09 (d, J=7.8 Hz, 1H), 6.11-6.13 (m, 2H), 5.66(dd, J=6.6, 3.0 Hz, 1H), 4.50-4.53 (m, 2H), 4.33-4.43 (m, 3H), 4.26 (s,2H), 4.14-4.16 (m, 2H), 3.95 (t, J=9.9 Hz, 1H), 3.82 (d, J=14.4 Hz, 1H),3.73 (dd, J=13.8, 6 Hz, 1H), 3.56 (t, J=10.2 Hz, 1H), 2.22 (s, 3H). ¹³CNMR (150 MHz, D₂O) δ 175.55, 175.05, 166.60, 152.20, 141.97, 102.99,94.62, 88.99, 83.50 (d, J=8.9 Hz), 74.06, 71.47, 71.06, 69.97, 65.33,61.35, 53.98 (d, J=8.4 Hz), 39.76, 22.42. HRMS (ESI) m/z calcd forC₁₉H₃₀N₄O₁₈P₂(M+H) 665.1109, found 665.1113.

Uridine5′-diphospho-2-acetamido-6-azidoacetamido-2,6-dideoxy-α-D-glucopyranoside(UDP-GlcNAc6NAcN₃, F5-13). UDP-GlcNAc6NAcN₃(F5-13) was synthesized fromUDP-GlcNAc6NH₂ (F5-10) using the same process as described above forUDP-GlcNAcN₃ (F5-6). UDP-GlcNAc6NAcN₃ (F5-13) was obtained as a whitesolid in 61% yield (21 mg). ¹H NMR (600 MHz, D₂O) δ 7.89 (d, J=7.8 Hz,1H), 5.91 (m, 2H), 5.43 (dd, J=6.6, 3.0 Hz, 1H), 4.32-4.29 (m, 2H),4.19-4.22 (m, 3H), 4.00 (s, 2H), 3.92-3.95 (m, 2H), 3.74 (t, J=10.2 Hz,1H), 3.54 (s, 1H), 3.34 (t, J=9.6 Hz, 1H), 2.01 (s, 3H). ¹³C NMR (150MHz, D₂O) δ 174.85, 170.93, 166.40, 151.92, 141.75, 104.99, 102.71,94.42, 88.71, 83.16 (J=8.9 Hz), 73.87, 71.27, 71.01, 70.71, 69.67,65.10, 53.72 (J=8.1 Hz), 51.80, 39.92, 22.14. HRMS (ESI) m/z calcd forC₁₉H₂₉N₇O₁₇P₂ (M+H) 690.1173, found 690.1180.

Uridine5′-diphospho-2-acetamido-6-phenylacetamido-2,6-dideoxy-α-D-glucopyranoside(UDP-GlcNAc6NAcPh, F5-14). UDP-GlcNAc6NAcPh F5-14 was synthesized fromUDP-GlcNAc6NH₂ F5-10 using the same way as described above forUDP-GlcNAcPh (F5-7). UDP-GlcNAc6NAcPh (F5-14) was obtained as a whitesolid in 86% yield (30 mg). ¹H NMR (600 MHz, D₂O) δ 7.87 (d, J=8.4 Hz,1H), 7.36-7.38 (m, 2H), 7.29-7.32 (m, 3H), 5.87-5.89 (m, 2H), 5.48 (dd,J=6.6, 2.4 Hz, 1H), 4.16-4.29 (m, 5H), 3.92-3.98 (m, 2H), 3.78 (t, J=9.6Hz, 1H), 3.53-3.65 (m, 4H), 3.30 (t, J=9.6 Hz, 1H), 2.05 (s, 3H). ¹³CNMR (150 MHz, D₂O) δ 175.37, 174.89, 166.26, 151.75, 141.64, 135.34,129.22, 129.05, 127.38, 102.66, 94.50, 88.93, 83.12 (J=8.6 Hz), 73.94,71.48, 71.06, 70.60, 69.51, 64.99, 53.79 (J=8.3 Hz), 42.43, 40.01,22.19. HRMS (ESI) m/z calcd for C₂₅H₃₄N₄O₁₇P₂(M+H) 725.1473, found725.1484.

Uridine5′-diphospho-2-acetamido-6-(1,1′-biphenyl-4-yl)-acetamido-2,6-dideoxy-α-D-glucopyranoside(UDP-GlcNAc6NAcPh₂, F5-15). UDP-GlcNAc6NAcPh₂ (F5-15) was synthesizedfrom UDP-GlcNAc6NH₂ using the same way as described above forUDP-GlcNAcPh₂ (F5-8). UDP-GlcNAc6NAcPh₂ (F5-15) was obtained as a whitesolid in 88% yield (35 mg). ¹H NMR (600 MHz, D₂O) δ 7.75 (d, J=7.8 Hz,1H), 7.63 (d, J=7.2 Hz, 2H), 7.60 (d, J=7.2 Hz, 2H), 7.44-7.45 (m, 2H),7.34-7.45 (m, 3H), 5.77-5.80 (m, 2H), 5.44 (dd, J=7.2, 3.6 Hz, 1H),4.03-4.18 (m, 5H), 3.89-3.96 (m, 2H), 3.75 (t, J=9.6 Hz, 1H), 3.49-3.63(m, 4H), 3.28 (t, J=9.0 Hz, 1H), 2.01 (s, 3H). ¹³C NMR (150 MHz, D₂O) δ175.17, 174.79, 166.28, 151.63, 141.34, 140.12, 139.31, 134.70, 129.74,129.18, 127.75, 127.30, 126.88, 102.44, 94.39, 88.87, 82.62 (d, J=8.7Hz), 73.93, 71.18, 70.54, 69.27, 64.80, 53.77 (d, J=8.4 Hz), 42.03,40.16, 22.09. HRMS (ESI) m/z calcd for C₃₁H₃₈N₄O₁₇P₂ (M+H) 801.1785,found 801.1807.

Results and Discussion

As shown in FIG. 4, three enzymes were used in one-pot to synthesizeUDP-GlcNAc and derivatives. The first enzyme was an N-acetylhexosamine1-kinase cloned from Bifidobacterium longum strain ATCC55813(NahK_ATCC55813) which showed promiscuous substrate specificity and wereable to use N-sulfated, 3-O-sulfated, or 6-O-sulfated GlcNAc andderivatives as substrates for the formation of GlcNAcα1-phosphatederivatives. The second enzyme was an N-acetylglucosamine-1-phosphateuridylyltransferase that we cloned from Pasteurella multocida strainP-1059 (ATCC15742) (PmGlmU). It catalyzes the reversible formation ofUDP-GlcNAc and pyrophosphate from UTP and GlcNAcα1-phosphate withtolerance on some substrate modifications. The third enzyme was aninorganic pyrophosphatase also cloned from Pasteurella multocida strainP-1059 (PmPpA) for hydrolyzing the pyrophosphate by-product formed todrive the reaction towards the formation of UDP-GlcNAc and derivatives.A recombinant NahK cloned from another strain of Bifidobacterium longum(NahK_JCM1217) was used in the synthesis of GlcNAc-1-phosphate,GalNAc-1-phosphate, and their derivatives. The purifiedHexNAc-1-phosphates were then used in a one-pot two-enzyme systemcontaining a commercially available inorganic pyrophosphatase (PpA) anda GlmU cloned from E. coli (EcGlmU) or an AGX1 cloned from human for thesynthesis of UDP-GlcNAc, dNDP-GlcNAc, dNDP-Glc, UDP-GalNAc, andderivatives. Nevertheless, chemoenzymatic synthesis of UDP-GlcNAcderivatives using all three enzymes in one-pot has not been reported. Inaddition, UDP-GlcNAc derivatives containing N-sulfated glucosamine orO-sulfated GlcNAc have not been synthesized using the combination ofthese three enzymes.

As shown in Table 5b, the one-pot three-enzyme system (FIG. 4) was quiteefficient in synthesizing UDP-GlcNAc (T5b-9, 81%), its C-2 derivativessuch as UDP-N-trifluoroacetylglucosamine (UDP-GlcNTFA, T5b-10, 97%) andUDP-2-azido-2-deoxy-glucose (UDP-GlcN₃, T5b-11, 54%), as well as its C-6derivatives including UDP-N-acetyl-6-azido-6-deoxy-glucosamine(UDP-GlcNAc6N₃, T5b-12, 72%) and UDP-N-acetyl-6-O-sulfo-glucosamine(UDP-GlcNAc6S, T5b-13, 62%) from GlcNAc (T5b-1) and derivatives(T5b-2-T5b-5). An interesting observation was that the yield of theone-pot three-enzyme reaction was improved from 81% to 97% when theN-acetyl group of GlcNAc was substituted by an N-trifluoroacetyl groupin GlcNTFA (T5b-2). However, while 6-O-sulfated GlcNAc (GlcNAc6S, T5b-5)was used as a substrate to produce UDP-GlcNAc6S (T5b-13) in 62% yield,the synthesis of its N-trifluoroacetyl analogue UDP-6-O-sulfo-GlcNTFA(UDP-GlcNTFA6S, T5b-14) from 6-O-sulfo-GlcNTFA (GlcNTFA6S, T5b-6) wasnot successful. In addition, although both 2-azido-2-deoxy-glucose(T5b-3) and 6-O-sulfo-GlcNAc (T5b-5) could be used for the synthesis ofthe corresponding UDP-GlcNAc derivatives UDP-GlcN₃ T5b-11 andUDP-GlcNAc6S T5b-13 in 54% and 62% yields, respectively, the synthesisof UDP-2-azido-2-deoxy-6-O-sulfo-glucose (UDP-GlcN₃6S, T5b15) fromGlcN₃6S (T5b-7) with the combined modifications at C-2 and C-6 was notsuccessful. Furthermore, the one-pot three-enzyme synthesis ofUDP-N-sulfo-glucosamine (UDP-GlcNS, T5b-16) from N-sulfo-glucosamine(GlcNS, T5b-8) was not achieved. As compounds T5b-3-T5b-8 have all beenshown to be weak substrates for NahK_ATCC55813, the successful synthesisof compounds T5b-11-T5b-13 and the unsuccessful synthesis of compoundsT5b-14-T5b-16 by the one-pot three-enzyme system indicate that thesubstrate specificity of PmGlmU is most likely the limiting factor.

TABLE 5b Synthesis of UDP-GlcNAc and its derivatives using the one-potthree- enzyme system shown in Figure 4. ND, not detected. YieldsSubstrates Products (%)

81

97

54

72

62

ND

ND

ND

Taking advantage of the substrate promiscuity of NahK_ATCC55813 andPmGlmU, UDP-GlcNAc and a number of its natural and non-naturalderivatives were synthesized efficiently by the one-pot three-enzymesystem illustrated in Scheme 1. However, the success of the approachrelied on the substrate promiscuity of all enzymes used. In order toincrease the size of the library of UDP-GlcNAc derivatives with variousmodifications that can be used to test the activity of diverseGlcNAc-transferases, we further carried out chemical diversification ofchemoenzymatically-produced UDP-GlcNAc derivatives.

The N-TFA group in UDP-GlcNTFA (T5b-10) as well as the N₃ group inUDP-GlcN₃ (T5b-11) UDP-GlcNAc6N₃ (T5b-12), and UDP-GlcN₃6S (T5b-15) canbe easily converted to a free amine, allowing further modifications togenerate a diverse array of N-substituted UDP-GlcNAc derivatives. Asshown in FIG. 5A, the N-TFA group at C2 of UDP-GlcNTFA T5b-10 (or F5-1)was removed under mild basic condition to produce UDP-glucosamine(UDP-GlcNH₂, F5-2) in 98% yield. Selective acylation of the free aminegroup in F5-2 using various acyl chlorides produced C-2 modifiedUDP-GlcNAc derivatives UDP-N-acetoxyacetylglucosamine (UDP-GlcNGcAc,F5-4), UDP-N-azidoacetylglucosamine (UDP-GlcNAz, F5-6),UDP-N-phenylacetylglucosamine (UDP-GlcNPh, F5-7), andUDP-N-(1,1′-biphenyl-4-yl)acetylglucosamine (UDP-GlcNPh₂, F5-8) in68-95% yields. Deacetylation of compound F5-4 using catalytic amount ofNaOMe in MeOH provided UDP-N-hydroxyacetylglucosamine (UDP-GlcNGc, F5-5)in 98% yield. In addition, although UDP-GlcNS T5b-16 (or F5-3) wasunable to be prepared from GlcNS (T5b-8) (Table 5b) in the one-potthree-enzyme system, it was readily obtained by N-sulfation of compoundF5-2 with Py.SO₃ in 2 M of NaOH aqueous solution in a very good yield(86%) (FIG. 5A). Similarly as shown in FIG. 5B, catalytic hydrogenationof the azido group at the C-6 of UDP-GlcNAc6N₃ T5b-12 (or F5-9)generated UDP-6-amino-6-deoxyl-N-acetylglucosamine (UDP-GlcNAc6NH₂,F5-10) with an excellent yield (96%). Selective acylation of the freeamino group of F5-10 using various acyl chlorides produced C-6 modifiedUDP-GlcNAc derivatives includingUDP-6-acetoxyacetamido-N-acetylglucosamine (UDP-GlcNAc6NGcAc, F5-11),UDP-6-azidoacetamido-N-acetylglucosamine (UDP-GlcNAc6NAz, F5-13),UDP-6-phenylacetamido-N-acetylglucosamine (UDP-GlcNAc6NPh, F5-14), andUDP-N-(1,1′-biphenyl-4-yl)acetamido-N-acetylglucosamine(UDP-GlcNAc6NPh₂, F5-15) in 61-91% yields. Finally, C-6 modifiedderivative UDP-N-hydroxyacetamido-N-acetylglucosamine (UDP-GlcNAc6NGc,F5-12) was obtained in 98% yield by treating compound F5-11 in NaOMe andmethanol.

Example 3 Preparation of UDP-GalNAc

One-Pot Three-Enzyme Synthesis of uridine5′-diphospho-2-acetamido-2-deoxy-α-D-glacopyranoside (UDP-GalNAc).GalNAc (100 mg, 1.0 eq), ATP (1.2 eq.), and UTP (1.2 eq.) were dissolvedin water in a 50 mL centrifuge tube containing Tris-HCl buffer (100 mM,pH 8.0) and MgCl₂ (10 mM). After the addition of NanK_ATCC55813 (3.5mg), PmGlmU (5 mg), and PmPpA (2.5 mg), water was added to bring thevolume of the reaction mixture to 20 mL. The reaction was carried out byincubating the solution in an isotherm incubator at 37° C. for 24 h withgentle shaking. Product formation was monitored by TLC(EtOAc:MeOH:H₂O=3:2:1 by volume) with p-anisaldehyde sugar staining. Thereaction was stopped by adding the same volume of ice-cold ethanol andincubating at 4° C. for 30 min. The mixture was concentrated and passedthrough a BioGel P-2 gel filtration column to obtain the desiredproduct. Silica gel column purification (EtOAc:MeOH:H₂O=4:2:1) wasapplied for further purification to give pure target compound. Yield,83% (228 mg); white foam. ¹H NMR (600 MHz, D₂O) δ 7.93 (d, J=8.4 Hz,1H), 5.94-5.96 (m, 2H), 5.55 (dd, J=6.6, 3.0 Hz, 1H), 4.27-4.36 (m, 2H),4.22-4.27 (m, 3H), 4.16-4.18 (m, 2H), 4.02 (d, J=3.0 Hz, 1H), 3.95 (dd,J=10.8, 3.0 Hz, 1H), 3.71-3.78 (m, 2H), 2.06 (s, 3H). ¹³C NMR (150 MHz,D₂O) δ 175.05, 166.31, 151.85, 141.89, 102.82, 94.75, 88.70, 83.03 (d,J=8.6 Hz), 73.97, 72.32, 69.79, 68.50, 67.64, 65.14, 61.17, 49.95 (d,J=7.8 Hz), 22.24. HRMS (ESI) m/z calcd for C₁₇H₂₈N₃O₁₇P₂ (M+H) 608.0894,found 608.0906.

Example 4 Preparation of UDP-Sugars Using Sugar-1-P Kinases, BLUSP, andPmPpA

General Methods for Compound Purification and Characterization.Chemicals were purchased and used without further purification. ¹H NMRand ¹³C NMR spectra were recorded on a Varian Mercury 600 NMRspectrometer. High resolution electrospray ionization (ESI) mass spectrawere obtained in negative mode using Thermo Electron LTQ-Orbitrap massspectrometer. Silica gel 60 Å (Sorbent Technologies) was used for flashcolumn chromatography. Thin-layer chromatography (TLC) was performed onsilica gel plates 60 GF254 (Sorbent Technologies) using anisaldehydesugar stain for detection. Gel filtration chromatography was performedwith a column (100 cm×2.5 cm) packed with BioGel P-2 Fine resins(Bio-Rad). GlcN₃ (T6-9) (Lau K, Thon, V, Yu H, Ding L, Chen Y, Muthana MM, Wong D, Huang R, and Chen X. Chem. Commun. 2010, 46, 6066-6068), ManF(T6-11) (Burkart M D, Zhang Z, Hung S-C, and Wong C-H. J. Am. Chem. Soc.1997, 119, 11743-11746; Cao H, Li Y, Lau K, Muthana S, Yu H, Cheng J,Chokhawala H A, Sugiarto G, Zhang L, and Chen X. Org. Biomol. Chem.2009, 7, 5137-5145), GalN₃ (T6-4) and ManN₃ (T6-14) (Yu H, Yu H, KarpelR, cand Chen X. Bioorg. Med. Chem. 2004, 12, 6427-6435) were previouslysynthesized using reported methods. NahK_ATCC15697, EcGalK, SpGalK, andPmPpA were overexpressed as described previously.

One-Pot Multienzyme Synthesis of UDP-Sugars. Monosaccharides andderivatives (30-100 mg, 1.0 eq.), ATP (1.2 eq.), and UTP (1.3 eq.) weredissolved in water in a 15 mL centrifuge tube containing Tris-HCl buffer(100 mM, pH 8.0) and MgCl₂ (10 mM). After the addition of appropriateamount of NahK_ATCC15697, EcGalK, or SpGalK (1.3-4.5 mg), BLUSP (1.0-2.5mg), and PmPpA (1.5-2.5 mg), millipore water was added to bring thetotal volume of the reaction mixture to 10 mL. The reaction was carriedout by incubating the solution in an isotherm incubator for 24 hr at 37°C. with gentle shaking or without shaking. In the synthesis of UDP-Glc,commercially available Glc-1-P (55.2 mg), UTP (1.2 eq.), Tris-HCl buffer(100 mM, pH 8.0), and MgCl₂ (10 mM) were used along with BLUSP (1 mg)and PmPpA (1.5 mg). The reaction was left for 2 hr at 37° C. in isothermwith gentle shaking. Product formation was monitored by TLC(EtOAc:MeOH:H₂O:AcOH=5:3:3:0.3 by volume) with p-anisaldehyde sugarstaining. The reaction was terminated by adding the same volume ofice-cold ethanol and incubating at 4° C. for 30 min followed bycentrifugation remove the enzymes. The supernatant was collected andconcentrated and passed through a BioGel P-2 gel filtration column toafford the product. Silica gel column purification(EtOAc:MeOH:H₂O=7:3:2) was applied when necessary to achieve furtherpurification.

Uridine 5′-diphospho-α-D-galactopyranoside (UDP-Gal, T6-16). 135 mg.Yield, 86%; white foam. ¹H NMR (600 MHz, D₂O) δ 7.93 (d, J=8.4 Hz, 1H),5.97-5.95 (m, 2H), 5.63 (dd, J=7.2, 3.6 Hz, 1H), 4.37-4.35 (m, 2H),4.28-4.18 (m, 3H), 4.16 (t, J=6 Hz, 1H), 4.02 (d, J=3 Hz, 1H), 3.90 (dd,J=10.2, 3.6 Hz, 1H), 3.80 (dt, J=10.2, 3.3 Hz, 1H), 3.76-3.71 (m, 2H).¹³C NMR (150 MHz, D₂O) δ 166.39, 151.96, 141.78, 102.80, 96.01 (d, J=6.6Hz), 88.65, 83.32 (d, J=8.9 Hz), 73.93, 72.11, 69.78, 69.43, 69.24,68.50 (d, J=7.8 Hz), 65.15 (d, J=5.0 Hz), 61.16. HRMS (ESI) m/z calcdfor C₁₅H₂₄N₂O₁₇P₂ (M−H) 565.0472, found 565.0453.

Uridine 5′-diphospho-α-D-glucopyranoside (UDP-Glc, T6-21). 82 mg. Yield,99%; white foam. ¹H NMR (600 MHz, D₂O) δ 7.94 (d, J=8.4 Hz, 1H),5.98-5.96 (m, 2H), 5.59 (dd, J=7.2, 3.6 Hz, 1H), 4.37-4.35 (m, 2H),4.28-4.18 (m, 3H), 3.9-3.83 (m, 2H), 3.78-3.74 (m, 2H), 3.53 (dt, J=9.6,3.3 Hz, 1H), 3.46 (t, J=9.6 Hz, 1H). ¹³C NMR (150 MHz, D₂O) δ 166.20,151.75, 141.52, 102.57, 95.51 (d, J=6.8 Hz), 88.35, 83.07 (d, J=8.9 Hz),73.67, 72.72 (2C), 71.45 (d, J=8.4 Hz), 69.52, 69.05, 64.86 (d, J=5.6Hz), 60.20. HRMS (ESI) m/z calcd for C₁₅H₂₄N₂O₁₇P₂ (M−H) 565.0472, found565.0458.

Uridine 5′-diphospho-2-deoxy-α-D-glucopyranoside (UDP-2-deoxyGlc,T6-22). 96 mg. Yield, 56%; white foam. ¹H NMR (600 MHz, D₂O) δ 7.95 (d,J=8.4 Hz, 1H), 5.96-5.95 (m, 2H), 5.70 (dd, J=7.2, 1.8 Hz, 1H),4.36-4.33 (m, 2H), 4.27-4.16 (m, 3H), 4.0-3.95 (m, 1H), 3.86-3.75 (m,3H), 3.39 (t, J=9.6 Hz, 1H), 2.28-2.24 (m, 1H), 1.74-1.68 (m, 1H), (150MHz, D₂O) δ 168.99, 154.54, 144.38, 105.38, 97.63 (d, J=5.7 Hz), 91.25,85.86 (d, J=9.0 Hz), 76.51, 76.13, 73.33, 72.32, 70.46, 67.63 (d, J=5.0Hz), 63.21, 40.18 (d, J=7.2 Hz), HRMS (ESI) m/z calcd for C₁₅H₂₄N₂O₁₆P₂(M−H) 549.0523, found 549.0513.

Uridine 5′-diphospho-2-amino-2-deoxy-α-D-glucopyranoside (UDP-GlcNH₂,T6-23). 56 mg. Yield, 43%; white foam. ¹H NMR (600 MHz, D₂O) δ 7.93 (d,J=7.8 Hz, 1H), 5.97-5.94 (m, 2H), 5.82 (d, J=6.0 Hz, 1H), 4.36-4.34 (m,2H), 4.28-4.17 (m, 3H), 3.92-3.90 (m 2H), 3.86 (dd, J=12.0, 2.4 Hz, 1H),3.81 (dd, J=12.6, 4.2 Hz, 1H), 3.55 (t, J=9.9 Hz, 1H), 3.37 (d, J=10.8Hz, 1H). ¹³C NMR (150 MHz, D₂O) δ 166.40, 151.93, 141.75, 102.71, 92.87,88.74, 83.21 (d, J=9 Hz), 73.91, 73.39, 69.85, 69.69, 69.16, 65.23,60.09, 54.27 (d, J=8.4 Hz). HRMS (ESI) m/z calcd for C₁₅H₂₅N₃O₁₆P₂ (M−H)564.0632, found 564.0619.

Uridine 5′-diphospho-2-azido-2-deoxy-α-D-glucopyranoside (UDP-GlcN₃,T6-24). 88 mg, Yield, 61%; white foam. ¹H NMR (600 MHz, D₂O) δ 7.95 (d,J=8.4 Hz, 1H), 5.96-5.95 (m, 2H), 5.67 (dd, J=7.2, 3 Hz, 1H), 4.36-4.33(m, 2H), 4.27-4.18 (m, 3H), 3.93-3.88 (m, 2H), 3.85-3.76 (m, 2H), 3.53(t, J=9.6 Hz, 1H), 3.38 (d, J=10.8 Hz, 1H). ¹³C NMR (150 MHz, D₂O) δ166.14, 151.75, 141.61, 102.79, 94.39 (d, J=4.5 Hz), 88.47, 83.48 (d,J=8.4 Hz), 73.68, 72.85, 70.61, 69.53, 69.24, 64.87, 62.71 (d, J=7.8Hz), 60.05. HRMS (ESI) m/z calcd for C₁₅H₂₃N₅O₁₆P₂ (M−H) 590.0537, found590.0524.

Uridine 5′-diphospho-α-D-mannopyranoside (UDP-Man, T6-26)

60 mg. Yield, 60%; white foam. ¹H NMR (600 MHz, D₂O) δ 7.93 (d, J=8.4Hz, 1H), 5.96-5.94 (m, 2H), 5.51 (d, J=7.2, 1H), 4.35-4.18 (m, 5H), 4.02(m, 1H), 3.89-3.82 (m, 3H), 3.75 (dd, J=12, 4.8 Hz, 1H), 3.67 (t, J=9.9Hz, 1H). ¹³C NMR (150 MHz, D₂O) δ 166.40, 151.94, 141.77, 102.79, 96.64(d, J=5.5), 88.70, 83.24 (d, J=8.7 Hz), 73.93, 73.91, 70.38 (d, J=9.3Hz), 69.98, 69.74, 66.56, 65.15 (d, J=4.7 Hz), 60.92. HRMS (ESI) m/zcalcd for C₁₅H₂₄N₂O₁₇P₂ (M−H) 565.0472, found 565.0467.

Uridine 5′-diphospho-2-fluoro-2-deoxy-α-D-mannopyranoside (UDP-ManF,T6-27). 142 mg. Yield, 92%; white foam. ¹H NMR (600 MHz, D₂O) δ 7.94 (d,J=8.4 Hz, 1H), 5.97-5.95 (m, 2H), 5.70 (t, J=6.3 Hz, 1H), 4.39-4.35 (m,2H), 4.36-4.33 (m, 2H), 4.28-4.16 (m, 3H), 4.00 (ddd, J=30.6, 9.6, 2.4Hz, 1H), 3.88-3.86 (m, 2H), 3.79 (d, J=12.6, 4.8 Hz, 1H), 3.74 (t, J=9.9Hz, 1H). ¹³C NMR (150 MHz, D₂O) δ 166.42, 151.98, 141.80, 102.84, 93.75(dd, J=31.2, 5.7 Hz), 89.75 (dd, J=173.6, 10.5 Hz), 88.75, 83.26 (d,J=9.0 Hz), 73.95, 73.84, 69.76, 69.32 (d, J=17.3 Hz), 66.46, 65.15 (d,J=5.1 Hz) 60.46. HRMS (ESI) m/z calcd for C₁₅H₂₃PN₂O₁₆P₂ (M−H) 567.0429,found 567.0426.

Uridine 5′-diphospho-2-azido-2-deoxy-α-D-mannopyranoside (UDP-ManN₃,T6-29). 259 mg, Yield, 90%; white foam. ¹H NMR (600 MHz, D₂O) δ 7.96 (d,J=8.4 Hz, 1H), 6.00-5.98 (m, 2H), 5.62 (d, J=7.2 Hz, 1H), 4.39-4.35 (m,2H), 4.31-4.18 (m, 3H), 4.16-4.13 (m, 2H), 3.87-3.83 (m, 2H), 3.77 (dd,J=12.6, 4.8 Hz, 1H), 3.70 (t, J=9.6 Hz, 1H). ¹³C NMR (150 MHz, D₂O) δ166.42, 151.97, 141.81, 102.84, 94.86 (d, J=5.7 Hz), 88.80, 83.24 (d,J=8.9 Hz), 73.96, 73.95, 70.09, 69.74, 66.49, 65.16 (d, J=5.0 Hz), 64.18(d, J=9.5 Hz) 60.64. HRMS (ESI) m/z calcd for C₁₅H₂₃N₅O₁₆P₂ (M−H)590.0537, found 590.0532.

Uridine 5′-diphospho-2-acetamido-2-deoxy-α-D-mannopyranoside(UDP-ManNAc, T6-30). Yield for two steps from UDP-ManN₃ (T6-29), 79%;white foam. ¹H NMR (600 MHz, D₂O) δ 7.96 (d, J=7.8 Hz, 1H), 5.98-5.95(m, 2H), 5.44 (dd, J=7.8, 1.8 Hz, 1H), 4.43 (dd, J=4.8, 1.8 Hz, 1H),4.37-4.34 (m, 2H), 4.28-4.22 (m, 2H), 4.19-4.15 (m, 1H), 4.11 (dd,J=10.2, 4.8 Hz, 1H), 3.90 (dt, J=10.2, 3.0 Hz, 1H), 3.85 (d, J=3.6 Hz,1H), 3.62 (t, J=10.2 Hz, 1H), 2.03 (s, 3H). ¹³C NMR (150 MHz, D₂O) δ175.59, 166.16, 151.75, 141.55, 102.57, 95.35, 88.23, 83.17 (d, J=8.9Hz), 73.73, 73.20, 69.58, 68.72, 66.29, 64.82, 59.23, 52.94 (d, J=8.9Hz), 21.85. HRMS (ESI) m/z calcd for C₁₇H₂₇N₃O₁₇P₂ (M−H) 606.0737, found606.0723.

TABLE 6 Synthesis of UDP-monosaccharides using the one-pot three-enzymesystem shown in Figure 9. ND, not detected. Yield Substrate KinaseProduct (%)

EcGalK or SpGalK

86

EcGalK or SpGalK

ND

EcGalK

ND

SpGalK

ND

SpGalK

ND

None

99

NahK

56

NahK

43

NahK

61

NahK

ND

NahK

60

NahK

92

NahK

ND

NahK

90

NahK

ND

We were able to identify at least one of these kinases for eachmonosaccharide that gave a yield higher than 58% for the formation ofthe corresponding monosaccharide-1-phosphate. The observed results fromthin-layer chromotography (TLC) and capillary electrophoresis (CE)(Table 7) confirmed the previously reported activities of NahK, SpGalK,and EcGalK toward their respective substrates except for mannosamine(T6-13), a NahK substrate which was not previously tested.

TABLE 7 Yields of the kinase reactions monitored by the conversion ofATP to ADP in capillary electrophoresis (CE) assays. ATP Conversion (%)Substrate Kinase 1 hr 4 hr 24 hr No No <2 <5   11.1 T6-1 Gal SpGalK 92.2NA NA T6-1 Gal EcGalK 90.3 NA NA T6-2 2-deoxyGal SpGalK 80.3 89.5 NAT6-2 2-deoxyGal EcGalK 78.5 87.6 NA T6-3 GalNH₂ EcGalK 90.2 NA NA T6-4GalN₃ SpGalK 45.7 79.0 81.2 T6-5 GalNAc SpGalK 11.8 24.7 69.5 Glc EcGalK8.2 13.0 66.4 Glc SpGalK 6.9 13.8 75.5 Glc NahK 10.3 18.6 82.2 T6-72-deoxyGlc NahK 36.8 69.6 79.4 T6-8 GlcNH₂ NahK 11.9 28.0 67.1 T6-9GlcN₃ NahK 12.4 25.9 71.2 T6-10 GlcNAc NahK 72.6 84.6 85.5 T6-11 ManNahK 29.6 69.3 75.1 T6-12 Man2F NahK 57.9 67.9 78.2 T6-13 ManNH₂ NahK10.3 22.8 58.0 T6-14 ManN₃ NahK 34.9 65.9 76.4 T6-15 ManNAc NahK 11.426.1 73.8 Abbreviation: NA, not assayed.

The synthesis of all other UDP-sugars in Table 6 was carried out usingthe one-pot three-enzyme system shown in FIG. 9. As shown in Table 6,the one-pot three-enzyme system provided excellent yields for theformation of UDP-Gal (T6-16, 86%), UDP-ManF (T6-27, 92%), and UDP-ManN₃(T6-29, 90%) from the corresponding monosaccharides Gal (T6-1), ManF(T6-12), and ManN₃ (T6-14), respectively. Three of the derivatives ofUDP-Glc including UDP-2-deoxyGlc (T6-22), UDP-GlcNH₂ (T6-23), andUDP-GlcN₃ (T6-24) were obtained from 2-deoxyGlc (T6-7), glucosamine(GlcNH₂, T6-8) and GlcN₃ (T6-9) in 56%, 43%, and 61% yields,respectively. The moderate yields for these three compounds may beattributed by less optimal NahK kinase activity for GlcNH₂ (T6-8) andGlcN₃ (T6-9), and the less optimal BLUSP activity for 2-deoxyGlc (T6-7).UDP-Man (T6-26) was synthesized from Man (T6-11) in moderate 60% yieldusing the one-pot three-enzyme system and the moderate yield was mostlikely due to the less optimal activity of BLUSP towards Man-1-P. Thesynthesis of four UDP-Gal derivatives including its 2-deoxy,2-deoxy-2-amido-, 2-deoxy-2-azido-, and 2-deoxy-2-acetamido-derivatives(T6-17-T6-20) using the one-pot three-enzyme system was not successful.In addition, UDP-GlcNAc (T6-25), UDP-ManNH₂ (T6-28), and UDP-ManNAc(T6-30) could not be produced from the corresponding monosaccharides(T6-10, T6-13, and T6-15) using the one-pot three-enzyme system. Thesewere most likely due to the substrate restriction of BLUSP instead ofkinases used.

Although UDP-ManNH₂ (T6-28) and UDP-ManNAc (T6-30) were not directlyavailable from ManNH₂ (T6-13) and ManNAc (T6-15), respectively, via theone-pot three-enzyme reaction shown in FIG. 9, they can be readilyprepared via simple chemical modification reactions from UDP-ManN₃(T6-29) obtained from the one-pot three-enzyme system. As shown in FIG.8, a simple one-step catalytic hydrogenation of UDP-ManN₃ (T6-29)produced UDP-ManNH₂ (T6-28). Acetylation of the amino group inUDP-ManNH₂ (T6-28) provided an easy access of UDP-ManNAc (T6-30). Thesimilar chemical acylation of UDP-ManNH₂ can be used to synthesize otheracyl derivatives of UDP-ManNAc.

Example 5 Synthesis of UDP-Uronic Acids Using AtGlcAK, BLUSP, and PmPpA

Mass Spectrometry Analysis of One-Pot Multienzyme Synthesis of UDP-GlcA,UDP-IdoA, and UDP-GalA. Enzymatic assays were carried out at in a totalvolume of 10 μL in Tris-HCl buffer (100 mM, pH 7.5) containing GlcA(GalA, or IdoA) (10 mM), ATP (20 mM), MgCl₂ (20 mM), and AtGlcAK (23μg). Reactions were allowed to proceed at 37° C. for 15 hr and monitoredusing thin-layer chromatographic analysis using n-PrOH:H₂O:NH₄OH=7:4:2(by volume) as a developing solvent. p-Anisaldehyde sugar stain followedby heating the TLC plates on hot plate was used for visualizingcompounds on the TLC plates. After 24 hr, BLUSP (5 μg), PmPpA (5 μg) andUTP (12 mM) were added to the reaction mixture. The reactions wereallowed to proceed at 37° C. for another 24 hr. The reactions were thenquenched with the same volume of 100% ethanol, centrifuged at 13000 rpmfor 2 min, and the reaction mixtures were stored at −20° C.

For LC-MS analysis, 2 μL of each sample was diluted 100-fold and 8 μLwas injected to a Waters spherisorb ODS-2 column (5 μm particles, 250 mmlength, 4.6 mm I.D.). Each sample was eluted with 30% acetonitrile inH₂O and detected by ESI-MS in the negative mode.

AtGlcAK was shown to be active on GlcA, GalA, and IdoA by TLC and LC-MSanalyses. One-pot three-enzyme strategy containing AtGlcAK, BLUSP, andPmPpA (FIG. 10) was shown to be able to produce UDP-GlcA, UDP-GalA, andUDP-IdoA from their corresponding monosaccharides GlcA, GalA, and IdoArespectively in small-scale assays confirmed by LC-MS or HRMS (FIG. 30).

Example 6 Preparation of GlcNAcα1-4GlcA Disaccharide Derivatives

General Methods for Compound Purification and Characterization.Chemicals were purchased and used without further purification. ¹H NMRand ¹³C NMR spectra were recorded on Varian VNMRS 600 MHz and BrukerAvance 800 MHz spectrometer. High resolution electrospray ionization(ESI) mass spectra were obtained at the Mass Spectrometry Facility inthe University of California, Davis. Silica gel 60 Å (SorbentTechnologies) was used for flash column chromatography. Analyticalthin-layer chromatography (Sorbent Technologies) was performed on silicagel plates using anisaldehyde sugar stain for detection. Gel filtrationchromatography was performed with a column (100 cm×2.5 cm) packed withBioGel P-2 Fine resins. ATP, UTP, GlcNAc, Glc-1-P, NAD⁺, andglucuronolactone were purchased from Sigma. GlcNTFA, GlcNAc6N₃,UDP-GlcNAc, UDP-GlcNAz, UDP-GlcNAc6NGc were synthesized as describedpreviously. NanK_ATCC55813, PmGlmU and PmPpA were overexpressed asreported.

Chemical Synthesis of GlcAβ2AAMe. GlcAβ2AAMe was synthesized as outlinedin FIG. 13.

Synthesis of F13-2. Glucuronolactone F13-1 (2.0 g, 11.3 mmol) wasdissolved in dry MeOH (12 mL) under N₂. To the solution, 20 mg of sodiummethoxide was added. The reaction was stirred at room temperature for 3hours, and then MeOH was removed in vacuo. The resulting syrup wasfurther dried under high-vacuum. The above product was dissolved inpyridine (10 mL) and acetic anhydride (8 mL) under 0° C. and N₂. Thereaction was stirred from 0° C. to room temperature overnight. Themixture was concentrated and purified by flash column chromatography(Hexane:EtOAc=1:1, by volume) to provide white solid F13-2 in 67% yield.β-isomer: ¹H NMR (600 MHz, CDCl₃) δ 5.76 (d, J=7.8 Hz, 1H), 5.30 (t,J=9.6 Hz, 1H), 5.25 (t, J=9.6 Hz, 1H), 5.13 (t, J=7.8 Hz, 1H), 4.17 (d,J=9.6 Hz, 1H), 3.73 (s, 3H), 2.10 (s, 3H), 2.03 (s, 6H), 2.02 (s, 3H).¹³C NMR (150 MHz, D₂O) δ 170.13, 169.65, 169.53, 168.61, 167.37, 88.90,70.51, 69.24, 69.07, 69.00, 53.17, 20.95, 20.79, 20.61, 20.55. α-isomer:¹H NMR (600 MHz, CDCl₃) δ 6.39 (d, J=3.6 Hz, 1H), 5.51 (t, J=10.2 Hz,1H), 5.22 (t, J=10.2 Hz, 1H), 5.12 (dd, J=10.2, 3.6 Hz, 1H), 4.41 (d,J=10.2 Hz, 1H), 3.74 (s, 3H), 2.15 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H),2.01 (s, 3H). ¹³C NMR (150 MHz, CDCl₃) δ 170.04, 169.56, 169.32, 168.98,166.95, 91.48, 73.11, 71.94, 70.27, 69.06, 53.18, 20.93, 20.72, 20.70,20.63.

Synthesis of methyl 2,3,4-tri-O-acetyl-D-glucopyranuronate F13-3. Methyl1,2,3,4-tetra-O-acetyl-D-glucopyranuronate F13-2 (1.2 g, 3.2 mmol) wasdissolved in dry DMF (10 mL) under N₂. To the solution, benzylamine(0.42 mL, 3.8 mmol) was added. The mixture was stirred at r.t. for 16hours. The solvent was removed under reduced pressure and the residuewas purified by flash chromatography (Hexane:EtOAc=1:2, by volume) toafford a white solid F13c in 84% yield. ¹H NMR (600 MHz, CDCl₃) δ5.50-5.55 (m, 1H), 5.11-5.27 (m, 1H), 4.85-4.91 (m, 1H), 5.39-4.56 (m,2H), 3.70-3.72 (m, 3H), 1.99-2.05 (m, 9H). ¹³C NMR (150 MHz, CDCl₃) δ170.68, 170.45, 170.34, 170.30, 169.94, 169.81, 168.77, 167.83, 95.59,90.36, 72.98, 72.66, 71.81, 70.97, 69.73, 69.61, 69.34, 68.12, 53.21,53.11, 20.85, 20.84, 20.77, 20.70, 20.66, 20.65.

Synthesis of methyl2,3,4-tetra-O-acetyl-1-O-(3-chloropropyl)-β-D-glucopyranuronate F13-5.Methyl 2,3,4-tri-O-acetyl-D-glucopyranuronate F13-3 (800 mg, 2.4 mmol)was dissolved in 8 mL dichloromethane. Trichloroacetonitrile (1.3 mL, 12mmol) was added under N₂. After cooling to 0° C.,1,8-diazabicyclo[5.4.0]undee-7-ene (1,8-DBU) was added in a drop-wisemanner until the color of sodium changes to brown. The reaction mixturewas allowed to stir for 1 h and the mixture was concentrated to afford asticky dark brown residue. The flash column chromatography(Hexane:EtOAc=3:2, by volume) gives an off-white product F13-4 in 88%yield. To the mixture of S4 (200 mg, 0.42 mmol) and MS 4 Å, 8 mLdichloromethane was added, followed by 3-chloropropanol (0.25 mL, 2.1mmol). The mixture was stirred for 30 min at room temperature under N₂.After cooling to 0° C., boron trifluoride ether complex (0.06 mL, 0.42mmol) was added drop-wisely. The reaction was stirred at 0° C. for 3hours. After the TLC showed the reaction is completed, the mixture wasfiltered and the filtrate was washed with saturated NaHCO₃. The organiclayer was evaporated to give a crude residue which was purified bysilica gel chromatography (Hexane:EtOAc=3:2, by volume) to provide theproduct F13-5 in 64% yield. ¹H NMR (600 MHz, CDCl₃) δ 5.17-5.28 (m, 2H),4.97-5.00 (dd, J=9.6, 7.8 Hz, 1H), 4.53-4.54 (d, J=7.8 Hz, 1H),4.02-4.04 (d, J=9.6 Hz, 1H), 3.99-4.01 (dd, J=9.6, 4.8 Hz, 1H), 3.27 (s,3H), 3.66-3.70 (m, 1H), 3.57-3.59 (m, 2H), 2.05-2.09 (m, 1H), 2.04 (s,3H), 2.01 (s, 3H), 2.00 (s, 3H), 1.91-1.95 (m 1H). ¹³C NMR (150 MHz,CDCl₃) δ 170.30, 169.59, 169.52, 167.37, 101.21, 72.78, 72.14, 71.33,69.62, 66.79, 53.13, 41.48, 32.29, 20.83, 20.82, 20.71.

Synthesis of methyl2,3,4-tetra-O-acetyl-1-O-(3-azidopropyl)-β-D-glucopyranuronate F13-6.Methyl 2,3,4-tetra-O-acetyl-1-O-(3-chloropropyl)-β-D-glucopyranuronateF13-5 (412 mg, 1.0 mmol) was dissolved in 10 mL of DMF. To the solution,sodium azide (325 mg, 5.0 mmol) was added. The reaction was stirred at65° C. overnight. The solvent was removed under reduced pressure and theresidue was purified by flash chromatography (Hexane:EtOAc=3:2, v/v) toafford a white solid in 92% yield. ¹H NMR (600 MHz, CDCl₃) δ 5.19-5.27(m, 2H), 4.99-5.02 (t, J=7.8 Hz, 1H), 4.54-4.55 (d, J=7.8 Hz, 1H),4.02-4.04 (d, J=9.6 Hz, 1H), 3.94-3.95 (m, 1H), 3.75 (s, 3H), 3.58-3.62(m, 1H), 3.32-3.39 (m, 2H), 2.04 (s, 3H), 2.01 (s, 3H), 1.78-1.89 (m2H). ¹³C NMR (150 MHz, CDCl₃) δ 170.27, 169.53, 169.40, 167.34, 101.01,72.82, 72.23, 71.39, 69.60, 66.92, 53.09, 48.10, 29.11, 20.81, 20.79,20.68.

Synthesis of 1-O-(3-azidopropyl)-β-D-glucopyranuronic acid F13-7. Methyl2,3,4-tetra-O-acetyl-1-O-(3-azidopropyl)-⊕-D-glucopyranuronate F13-6(350 mg, 0.84 mmol) was dissolved in 5 mL MeOH. To the solution, sodiummethoxide was added until the pH go to 9.5. The reaction was stirred atroom temperature for 1 hr. After the TLC showed the reaction iscompleted, potassium hydroxide (60 mg, 2.52 mmol) and 10 mL water wasadded. After stirred at r.t. for 3 hours, the mixture was neutralizedwith DOWEX HCR-W2 (Fr) resin, filtered, and concentrated. The residuewas purified by flash column chromatography (EtOAc:MeOH:H₂O=6:2:1, byvolume) to afford white solid F13-7 in 79% yield. ¹H NMR (600 MHz, D₂O)δ 4.45-4.47 (d, J=7.8 Hz, 1H), 3.95-3.99 (m, 1H), 3.81-3.82 (d, J=9.0Hz, 1H), 3.71-3.75 (m, 1H), 3.49-3.54 (m, 2H), 3.42-3.45 (t, J=6.6 Hz,2H), 3.28-3.31 (t, J=8.4 Hz, 1H), 1.86-1.91 (m, 2H). ¹³C NMR (150 MHz,D₂O) δ 175.17, 102.36, 75.91, 75.64, 73.06, 71.86, 67.57, 48.05, 28.39.

Synthesis of GlcAβAAMe (F13-8). 1-O-(3-Azidopropyl)-β-D-glucopyranuronicacid F13-7 (100 mg, 0.44 mmol) was dissolved in 10 mL MeOH and 20 mg ofPd/C was added. The mixture was shaken under H₂ gas (4 Bar) for 1 h,filtered, and concentrated. The residue was further dried inhigh-vacuum. To a solution of the amine residue in 10 mL of DMF-MeOH(1:1, v/v), dry triethylamine (61 μL) was added under N₂. Then2-(methoxycarbonyl) succinanilic acid NHS ester³ (2AA-OSu, 306 mg, 0.88mmol) was added at 0° C. The reaction mixture was stirred at roomtemperature for overnight. The reaction mixture was concentrated and theresidue was purified by silica gel chromatography (EtOAc:MeOH:H₂O=8:2:1,by volume) to afford white solid GlcAβ2AAMe (F13-8) in 83% yield. ¹H NMR(600 MHz, D₂O) δ 7.88-7.89 (d, J=8.4 Hz, 1H), 7.79-7.80 (d, J=7.8 Hz,1H), 7.48-7.51 (t, J=7.8 Hz, 1H), 7.12-7.15 (t, J=7.8 Hz, 1H), 4.27-4.29(d, J=7.8 Hz, 1H), 3.83-3.86 (m, 1H), 3.81 (s, 3H), 3.58-3.60 (d, J=9.6Hz, 1H), 3.53-3.57 (m, 1H), 3.40-3.47 (m, 2H), 3.24-3.30 (m, 2H),3.18-3.22 (m, 1H), 2.61-2.64 (t, J=7.2 Hz, 2H), 2.52-2.54 (t, J=7.2 Hz,2H), 1.72-1.77 (m, 2H). ¹³C NMR (150 MHz, D₂O) δ 175.17, 174.18, 172.66,168.75, 137.89, 134.16, 130.81, 124.41, 121.83, 118.36, 101.96, 75.66,75.46, 72.83, 71.68, 67.35, 52.63, 36.04, 32.61, 30.83, 28.23.

One-Pot Four-Enzyme Synthesis of Disaccharides F18a-F18c. As shown inFIG. 18A, GlAβ2AAMe (F13-8) (5 to 30 mg, 1 eq.), glucosamine derivatives(1.5 eq.), ATP (1.8 eq.), and UTP (1.8 eq.) were dissolved in water in a15 mL centrifuge tube containing Tris-HCl buffer (100 mM, pH 7.5) or MESbuffer (100 mM, pH 6.5) and MgCl₂ (10 mM). After the addition ofappropriate amount of NanK_ATCC55813 (0.5-2.1 mg), PmGlmU (1-3 mg),PmPpA (0.5-1.5 mg), and PmHS2 (1-6 mg), water was added to bring theconcentration of GlAβ2AAMe (F13-8) to 5 mM. The reaction was carried outby incubating the solution in an isotherm incubator for 12-36 h at 37°C. with gentle shaking. Product formation was monitored by TLC(EtOAc:MeOH:H₂O=4:2:1 by volume) with p-anisaldehyde sugar staining. Thereaction was stopped by adding the same volume of ice-cold ethanol andincubating at 4° C. for 30 min. The mixture was concentrated and passedthrough a BioGel P-2 gel filtration column to obtain the desiredproduct. Silica gel column purification (EtOAc:MeOH:H₂O=5:2:1) wasapplied when necessary to achieve further purification.

GlcNAcα1-4GlcAβ2AAMe F18-1. Yield: 95%; white foam. ¹H NMR (600 MHz,D₂O) δ 7.95-7.96 (d, J=8.4 Hz, 1H), 7.81-7.83 (d, J=8.4 Hz, 1H),7.61-7.64 (t, J=7.8 Hz, 1H), 7.29-7.32 (t, 0.1=8.4 Hz, 1H), 5.38-5.39(d, J=3.6 Hz, 1H), 4.32-4.33 (d, 0.1=7.8 Hz, 1H), 3.89 (s, 3H),3.79-3.88 (m, 4H), 3.70-3.73 (m, 4H), 3.60-3.63 (m, 1H), 3.55-3.58 (m,1H), 3.45-3.48 (t, J=9.6 Hz, 1H), 3.20-3.32 (m, 3H), 2.73-2.75 (t, J=6.6Hz, 2H), 2.58-2.61 (t, J=7.2 Hz, 2H), 2.04 (s, 3H), 1.73-1.78 (m, 2H).¹³C NMR (150 MHz, D₂O) δ 175.15, 174.61, 174.51, 173.61, 169.24, 137.16,134.12, 130.99, 125.45, 123.57, 121.10, 102.21, 96.98, 76.96, 76.69,75.90, 73.56, 72.01, 70.86, 69.80, 67.58, 60.22, 53.83, 52.93, 36.23,32.59, 31.20, 28.40, 22.06. HRMS (ESI) m/z calcd for C₂₉H₄₂N₃O₁₆ (M+H)688.2560, found 688.2563.

GlcNTFAα1-4GlcAβ2AAMe F18-2. Yield: 84%; white foam. ¹H NMR (600 MHz,D₂O) δ 7.95-7.97 (d, J=7.8 Hz, 1H), 7.88-7.89 (d, J=7.8 Hz, 1H),7.62-7.65 (t, J=7.8 Hz, 1H), 7.30-7.32 (t, J=7.8 Hz, 1H), 5.50-5.51 (d,J=3.6 Hz, 1H), 4.34-4.35 (d, J=7.8 Hz, 1H), 4.02-4.04 (dd, J=10.8, 4.2Hz, 1H), 3.91 (s, 3H), 3.83-3.88 (m, 4H), 3.75-3.77 (m, 3H), 3.63-3.66(m, 1H), 3.57-3.61 (m, 1H), 3.51-3.55 (t, J=9.6 Hz, 1H), 3.23-3.34 (m,3H), 2.74-2.76 (t, J=6.6 Hz, 2H), 2.61-2.63 (t, J=7.2 Hz, 2H), 1.77-1.81(m, 2H). ¹³C NMR (150 MHz, D₂O) δ 175.15, 174.63, 173.50, 169.26, 159.45(q, J=37.6 Hz), 137.49, 134.28, 131.09, 125.33, 123.27, 120.52, 117.02(q, J=284.7 Hz), 102.29, 96.51, 76.90, 76.73, 76.13, 73.61, 72.21,70.40, 69.85, 67.73, 60.32, 54.54, 53.00, 36.34, 32.75, 31.26, 28.51.HRMS (ESI) m/z calcd for C₂₉H₃₉F₃N₃O₁₆ (M+H) 742.2277, found 742.2284.

GlcNAc6N₃α1-4GlcAβ2AAMe F18-3. Yield: 89%; white foam. ¹H NMR (600 MHz,D₂O) δ 7.97-7.98 (d, J=7.8 Hz, 1H), 7.82-7.84 (d, J=8.4 Hz, 1H),7.63-7.66 (t, J=7.2 Hz, 1H), 7.32-7.34 (t, J=7.2 Hz, 1H), 5.40-5.41 (d,J=3.6 Hz, 1H), 4.34-4.35 (d, J=7.8 Hz, 1H), 3.91 (s, 3H), 3.83-3.90 (m,3H), 3.70-3.73 (m, 3H), 3.57-3.63 (m, 4H), 3.47-3.51 (t, J=9.6 Hz, 1H),3.22-3.33 (m, 3H), 2.74-2.77 (t, J=6.6 Hz, 2H), 2.60-2.62 (t, J=7.2 Hz,2H), 2.05 (s, 3H), 1.75-1.79 (m, 2H). ¹³C NMR (150 MHz, D₂O) δ 175.06,174.64, 174.53, 173.69, 169.28, 137.07, 134.10, 130.99, 125.54, 123.75,121.38, 102.21, 97.07, 76.88, 76.80, 76.04, 73.57, 70.85, 70.65, 70.44,67.60, 53.76, 52.94, 50.62, 36.26, 32.58, 31.23, 28.41, 22.07. HRMS(ESI) m/z calcd for C₂₉H₄₁N₆O₁₅ (M+H) 713.2625, found 713.2630.

Chemical Derivatization of GlcNTFAα1-4GlcAβ2AAMe (F18-2) to formdisaccharide GlcNH₂α1-4GlcAβ2AA (F24-9). DisaccharideGlcNTFAα1-4GlcAβ2AAMe (F18-2) (20 mg, 0.027 mmol) was dissolved in 8 mLof H₂O. The pH of the solution was adjusted to 10 by adding 1 N NaOH.After being vigorously stirred at r.t. for 1.5 hr, the reaction mixturewas neutralized with DOWEX HCR-W2 (H⁺) resin, filtered and concentrated.The residue was purified by flash column chromatography(EtOAc:MeOH:H₂O=3:2:1, by volume) to obtain a white solidGlcNH₂α1-4GlcAβ2AA (F24-9) in 86% yield. ¹H NMR (600 MHz, D₂O) δ8.14-8.15 (d, J=8.4 Hz, 1H), 7.87-7.88 (d, J=7.8 Hz, 1H), 7.49-7.51 (t,J=7.8 Hz, 1H), 7.20-7.22 (t, J=6.6 Hz, 1H), 5.65-5.66 (d, J=3.0 Hz, 1H),4.26-4.27 (d, J=7.8 Hz, 1H), 3.72-3.85 (m, 8H), 3.66-3.67 (t, J=8.4 Hz,1H), 3.46-3.52 (m, 2H), 3.20-3.35 (m, 3H), 2.74-2.76 (t, J=6.6 Hz, 2H),2.61-2.63 (t, J=6.6 Hz, 2H), 1.73-1.74 (m, 2H). ¹³C NMR (150 MHz, D₂O) δ175.15, 174.77, 174.68, 173.01, 137.67, 131.91, 130.82, 125.32, 124.25,120.95, 102.28, 97.77, 76.77, 76.51, 76.32, 73.27, 72.32, 69.60, 67.61,61.37, 60.24, 54.95, 36.21, 33.50, 31.57, 28.43. HRMS (ESI) m/z calcdfor C₂₆H₃₈N₃O₁₅ (M+H) 632.2303, found 632.2321.

PmHS2-Catalyzed Synthesis of Disaccharides F18-4-F186. As shown in FIG.18B, GlAβ2AAMe (F13-8) (5 to 10 mg, 1 eq.) and UDP-GlcNAc derivatives(1.2 eq.) were dissolved in water in a 15 mL centrifuge tube containingTris-HCl buffer (100 mM, pH 7.5) and MgCl₂ (10 mM). After the additionof appropriate amount PmHS2 (1-2 mg), water was added to bring thevolume of the reaction mixture to 10 mL. The reaction was carried out byincubating the solution in an isotherm incubator for 12 to 36 h at 37°C. with gentle shaking. Product formation was monitored by TLC(EtOAc:MeOH:H₂O=4:2:1 by volume) with p-anisaldehyde sugar staining. Thereaction was stopped by adding the same volume of ice-cold ethanol andincubating at 4° C. for 30 min. The mixture was concentrated and passedthrough a BioGel P-2 gel filtration column to obtain the desiredproduct. Silica gel column purification (EtOAc:MeOH:H₂O=5:2:1) wasapplied when necessary to achieve further purification.

GlcNGcα1-4GlcAβ2AAMe F18-4. Yield: 92%; white foam. ¹H NMR (600 MHz,D₂O) δ 7.97-7.98 (d, J=7.8 Hz, 1H), 7.81-7.82 (d, J=8.4 Hz, 1H),7.62-7.65 (t, J=7.2 Hz, 1H), 7.31-7.34 (t, J=8.4 Hz, 1H), 5.39-5.40 (d,J=3.6 Hz, 1H), 4.32-4.33 (d, J=7.8 Hz, 1H), 4.13 (s, 2H), 3.94-3.96 (dd,J=10.8, 4.2 Hz, 1H), 3.90 (s, 3H), 3.77-3.86 (m, 4H), 3.71-3.75 (m, 3H),3.61-3.64 (m, 1H), 3.55-3.59 (m, 1H), 3.48-3.51 (t, J=9.6 Hz, 1H),3.20-3.30 (m, 3H), 2.74-2.76 (t, J=6.6 Hz, 2H), 2.59-2.61 (t, J=7.2 Hz,2H), 1.74-1.78 (m, 2H). ¹³C NMR (150 MHz, D₂O) δ 175.18, 175.12, 174.62,173.67, 169.26, 137.05, 134.08, 130.97, 125.52, 123.72, 121.35, 102.19,97.05, 76.93, 76.66, 76.03, 73.48, 72.06, 70.81, 69.72, 67.57, 61.03,60.17, 53.45, 52.92, 36.22, 32.56, 31.20, 28.38. HRMS (ESI) m/z calcdfor C₂₉H₄₂N₃O₁₇ (M+H) 704.2509, found 704.2516.

GlcNAzα1-4GlcAβ2AAMe F18-5. Yield: 91%; white foam. ¹H NMR (600 MHz,D₂O) δ 7.97-7.99 (d, J=7.8 Hz, 1H), 7.82-7.83 (d, J=7.8 Hz, 1H),7.64-7.66 (t, J=7.2 Hz, 1H), 7.33-7.35 (t, J=7.8 Hz, 1H), 5.41-5.42 (d,J=3.6 Hz, 1H), 4.33-4.34 (d, J=7.8 Hz, 1H), 4.08 (s, 2H), 3.95-3.97 (dd,J=7.8, 3.6 Hz, 1H), 3.91 (s, 3H), 3.82-3.86 (m, 1H), 3.73-3.80 (m, 6H),3.62-3.65 (m, 1H), 3.56-3.60 (m, 1H), 3.48-3.51 (t, J=9.0 Hz, 1H),3.22-3.33 (m, 3H), 2.75-2.77 (t, J=6.6 Hz, 2H), 2.60-2.62 (t, J=6.6 Hz,2H), 1.75-1.79 (m, 2H). ¹³C NMR (150 MHz, D₂O) δ 175.15, 174.64, 173.71,170.83, 169.28, 137.04, 134.09, 130.98, 125.56, 123.77, 121.44, 102.21,96.88, 76.90, 76.70, 75.99, 73.52, 72.06, 70.72, 69.77, 67.60, 60.20,53.87, 52.93, 51.93, 36.24, 32.57, 31.23, 28.40. HRMS (ESI) m/z calcdfor C₂₉H₄₁N₆O₁₆ (M+H) 729.2574, found 729.2582.

GlcNAc6NGcα1-4GlcAβ2AAMe F18-6. Yield: 74%; white foam. ¹H NMR (600 MHz,D₂O) δ 7.98-7.99 (d, J=7.8 Hz, 1H), 7.82-7.83 (d, J=7.8 Hz, 1H),7.64-7.66 (t, J=7.8 Hz, 1H), 7.33-7.35 (t, J=7.8 Hz, 1H), 5.31-5.32 (d,J=3.6 Hz, 1H), 4.33-4.35 (d, J=8.4 Hz, 1H), 4.12 (s, 2H), 3.91 (s, 3H),3.81-3.90 (m, 4H), 3.68-3.74 (m, 3H), 3.56-3.63 (m, 3H), 3.50-3.53 (dd,J=13.8, 2.4 Hz, 1H), 3.22-3.32 (m, 3H), 2.76-2.78 (t, J=6.6 Hz, 2H),2.60-2.63 (t, J=7.2 Hz, 2H), 2.04 (s, 3H), 1.75-1.79 (m, 2H). ¹³C NMR(150 MHz, D₂O) δ 175.48, 175.29, 174.65, 174.55, 173.71, 169.28, 137.03,134.09, 130.99, 125.57, 123.81, 121.48, 102.16, 97.49, 77.08, 76.86,76.73, 73.57, 71.51, 70.72, 70.51, 67.57, 61.18, 53.77, 52.94, 39.62,36.24, 32.57, 31.24, 28.41, 22.08. HRMS (ESI) m/z calcd for C₃₁H₄₅N₄O₁₇(M+H) 745.2780, found 745.2787.

Example 7 Preparation of Trisaccharide Derivatives

Small Scale One-Pot Three-Enzyme Synthesis of Trisaccharides F20-1-F20-6by HPLC and MALDI-TOF MS Analysis. As shown in FIG. 19, Typicalenzymatic assays were performed in a total volume of 20 μL in Tris-HClbuffer (100 mM, pH 7.5) containing MgCl₂ (10 mM), UTP (7.5 mM),disaccharides (5 mM), Glc-1-P (6 mM), NAD⁺ (12 mM), GalU (2.5 μg), PmUgd(8 μg) and PmHS2 (11.5 μg). Reactions were allowed to proceed for 12 hrat 37° C. and quenched by adding ice-cold ethanol (20 μL) and water(1.96 mL) to make 100-fold dilution. The samples were then kept on iceuntil aliquots of 5 μL were injected and analyzed by a Shimadzu LC-2010Asystem equipped with a membrane on-line degasser, a temperature controlunit (maintained at 30° C. throughout the experiment), and afluorescence detector. A reverse phase Premier C18 column (250×4.6 mmI.D., 5 μm particle size, Shimadzu) protected with a C18 guard columncartridge was used. The mobile phase was 10% acetonitrile. Thefluorescent compounds 2AA derivatives were detected by excitation at 305nm and emission at 415 nm. The MS data of the products were acquiredusing MALDI Mass. See Table 8.

TABLE 8 HPLC and MALDI-TOF MS analysis data for the synthesis oftrisaccharides F20-1-F20-6 Starting Retention Product Retention Cal.Mass Measured Mass* Material Time (min) (Yield) Time (min) M + Na⁺ M +Na⁺ − H⁺ M + 2Na⁺ − 2H⁺ M + 3Na⁺ − 3H⁺ Compound 8.8 Compound 4.7885.2627 885.4562 907.4189 929.2829 F18-1 F20-1 (100%) Compound 13.5Compound 4.8 940.2423 939.3854 961.3454 983.3108 F18-2 F20-2 (72%)Compound 9.3 Compound 3.9 911.277 910.4507 932.4137 954.3776 F18-3 F20-3(100%) Compound 8.1 Compound 4.9 902.2655 901.4351 923.3950 945.3587F18-4 F20-4 (75%) Compound 10.5 Compound 4.8 927.2719 926.4009 948.3610970.3205 F18-5 F20-5 (95%) Compound 9.2 Compound 5.1 943.2920 942.4578964.4117 — F18-6 F20f (14%) *Measured values represent M + Na⁺, M + 2Na⁺− H⁺, M + 3Na⁺ − 2H⁺.

Preparative-Scale Preparation of TrisaccharideGlcAβ1-4GlcNTFAα1-4GlcAβ2AAMe F20-2 in a One-Pot Three-Enzyme System asShown in FIG. 19.

Disaccharide GlcNTFAα1-4GlcAβAAMe F18-2 (30 mg, 1 eq.), Glc-1-P (1.2eq), UTP (1.5 eq) and NAD⁺ (2.4 eq.) were dissolved in water in a 15 mLcentrifuge tube containing Tris-HCl buffer (100 mM, pH 7.0) and MgCl₂(10 mM). After the addition of appropriate amount of GalU (1 mg), PmUgd(3 mg), PmHS2 (4.5 mg), water was added to bring the volume of thereaction mixture to 8 mL. The reaction was carried out by incubating thesolution in an isotherm incubator at 37° C. for 12 hr with gentleshaking. Product formation was monitored by TLC (EtOAc:MeOH:H₂O=3:2:1 byvolume) with p-anisaldehyde sugar staining. The reaction was stopped byadding the same volume of ice-cold ethanol and incubating at 4° C. for30 min. The mixture was concentrated and passed through a BioGel P-2 gelfiltration column to obtain the desired product. The trisaccharide wasfurther purified by silica gel column chromatography(EtOAc:MeOH:H₂O=4:2:1) to obtain white solid trisaccharideGlcAβ1-4GlcNTFAα1-4GlcAβ2AAMe F20-2 in 87% yield. ¹H NMR (600 MHz, D₂O)δ 7.96-7.97 (d, J=7.8 Hz, 1H), 7.80-7.82 (d, J=8.4 Hz, 1H), 7.61-7.64(t, J=7.8 Hz, 1H), 7.31-7.33 (t, J=7.2 Hz, 1H), 5.44-5.45 (d, J=3.6 Hz,1H), 4.94-4.51 (d, J=7.8 Hz, 1H), 4.30-4.31 (d, J=7.8 Hz, 1H), 3.99-4.01(dd, J=11.4, 3.6 Hz, 1H), 3.94-3.97 (m, 1H), 3.90 (s, 3H), 3.80-3.85 (m,4H), 3.70-3.75 (m, 4H), 3.57-3.60 (m, 1H), 3.53-3.56 (m, 1H), 3.48-3.52(m, 2H), 3.35-3.37 (t, J=7.8 Hz, 1H), 3.20-3.31 (m, 3H), 2.73-2.76 (t,J=7.2 Hz, 2H), 2.58-2.61 (t, J=7.2 Hz, 2H), 1.73-1.77 (m, 2H). ¹³C NMR(150 MHz, D₂O) δ 174.88 (2C), 174.40, 173.45, 169.04, 159.11 (q, f=37.7Hz), 136.81, 133.86, 130.75, 125.31, 123.50, 121.14, 116.69 (q, J=284.6Hz), 102.27, 101.95, 96.01, 78.16, 76.60, 76.50, 76.02, 75.75, 75.05,73.31, 72.82, 71.68, 70.58, 68.59, 67.36, 59.22, 53.85, 52.70, 36.00,32.34, 30.99, 28.16. HRMS (ESI) m/z calcd for C₃₅H₄₇F₃N₃O₂₂ (M+H)918.2603, found 918.2613.

Preparative-Scale Preparation of TrisaccharideGlcAβ1-4GlcNH₂α1-4GlcAβ2AA (F24-11) in a One-Pot Three-Enzyme System asShown in FIG. 19.

Disaccharide GlcNH₂α1-4GlcAβ2AA (F24-9) (15 mg, 1 eq.), Glc-1-P (1.2eq), UTP (1.5 eq), and NAD⁺ (2.4 eq.) were dissolved in water in a 15 mLcentrifuge tube containing Tris-HCl buffer (100 mM, pH 7.0) and MgCl₂(10 mM). After the addition of appropriate amount of GalU (0.5 mg),PmUgd (1.5 mg), PmHS2 (2.5 mg), water was added to bring the volume ofthe reaction mixture to 4 mL. The reaction was carried out by incubatingthe solution in an isotherm incubator at 37° C. for 12 hr with gentleshaking. Product formation was monitored by TLC (EtOAc:MeOH:H₂O=3:2:1 byvolume) with p-anisaldehyde sugar staining. The reaction was stopped byadding the same volume of ice-cold ethanol and incubating at 4° C. for30 min. The mixture was concentrated and passed through a BioGel P-2 gelfiltration column to obtain the desired product. The trisaccharide wasfurther purified by silica gel column chromatography(EtOAc:MeOH:H₂O=3:2:1) to obtain white solid GlcAβ1-4GlcNH₂α1-4GlcAβ2AA(F24-11) in 84% yield. ¹H NMR (600 MHz, D₂O) δ 7.96-7.97 (d, J=6.6 Hz,1H), 7.81-7.82 (d, J=6.6 Hz, 1H), 7.62-7.65 (t, J=6.6 Hz, 1H), 7.31-7.34(d, J=6.6 Hz, 1H), 5.63-5.64 (d, J=3.6 Hz, 1H), 4.49-4.50 (d, J=6.6 Hz,1H), 4.35-4.37 (d, J=7.8 Hz, 1H), 3.9-3.98 (t, J=9.0 Hz, 1H), 3.91 (s,3H), 3.82-3.88 (m, 4H), 3.67-3.78 (m, 6H), 3.49-3.59 (m, 3H), 3.28-3.38(m, 3H), 3.21-3.25 (m, 1H), 2.74-2.76 (t, J=7.2 Hz, 2H), 2.60-2.62 (t,J=6.6 Hz, 2H), 1.75-1.77 (m, 2H). ¹³C NMR (150 MHz, D₂O) δ 175.82,175.08, 174.65, 173.66, 169.27, 137.14, 134.14, 131.01, 125.51, 123.66,121.23, 102.52, 102.23, 97.66, 78.32, 76.76, 76.61, 76.51, 75.90, 75.33,73.20, 73.09, 71.93, 70.99, 67.62, 59.48, 58.73, 54.66, 52.95, 36.24,32.60, 31.23, 28.41. HRMS (ESI) m/z calcd for C₃₂H₄₆N₃O₂₁ (M+H)807.2546, found 807.2557.

Example 8 Preparation of Tetrasaccharides

One-Pot Four-Enzyme Synthesis of TetrasaccharideGlcNAc6N₃α1-4GlcAβ1-4GlcNTFAcα1-4GlcAβ2AAMe F21-1. TrisaccharideGlcAβ1-4GlcNTFAα1-4GlcAβ2AAMe F20-2 (30 mg, 1 eq.), GlcNAc6N₃ (1.5 eq.),ATP (1.8 eq.), and UTP (1.8 eq.) were dissolved in water in a 15 mLcentrifuge tube containing MES buffer (100 mM, pH 6.5) and MgCl₂ (10mM). After the addition of appropriate amount of NanK_ATCC55813 (2.5mg), PmGlmU (3 mg), PmPpA (1.5 mg), and PmHS2 (4 mg), water was added tobring the volume of the reaction mixture to 6.5 mL. The reaction wascarried out by incubating the solution in an isotherm incubator for 18 hat 37° C. with gentle shaking. Product formation was monitored by TLC(EtOAc:MeOH:H₂O=4:2:1 by volume) with p-anisaldehyde sugar staining. Thereaction was stopped by adding the same volume of ice-cold ethanol andincubating at 4° C. for 30 min. The mixture was concentrated and passedthrough a BioGel P-2 gel filtration column to obtain the desiredproduct. The tetrasaccharide was further purified by silica gel columnchromatography (EtOAc:MeOH:H₂O=5:2:1) to obtain white solidtetrasaccharide GlcNAc6N₃α1-4GlcAβ1-4GlcNTFAα1-4GlcAβ2AAMe F21-1 in 93%yield. ¹H NMR (600 MHz, D₂O) δ 7.94-7.96 (d, J=7.8 Hz, 1H), 7.81-7.83(d, J=8.4 Hz, 1H), 7.60-7.63 (t, J=7.8 Hz, 1H), 7.29-7.31 (t, J=7.2 Hz,1H), 5.43-5.44 (d, J=3.6 Hz, 1H), 5.40-5.41 (d, J=4.2 Hz, 1H), 4.47-4.49(d, J=7.8 Hz, 1H), 4.29-4.31 (d, J=8.4 Hz, 1H), 3.93-4.00 (m, 2H),3.88-3.90 (m, 4H), 3.78-3.86 (m, 6H), 3.66-3.75 (m, 6H), 3.61-3.62 (d,J=2.4 Hz, 2H), 3.57-3.60 (m, 114), 3.53-3.56 (m, 1H), 3.45-3.48 (t,J=9.0 Hz, 1H), 3.34-3.36 (t, J=7.8 Hz, 1H), 3.19-3.30 (m, 3H), 2.72-2.74(t, J=6.6 Hz, 2H), 2.57-2.60 (t, J=6.6 Hz, 2H), 2.03 (s, 3H), 1.72-1.76(m, 2H). ¹³C NMR (150 MHz, D₂O) δ 174.85, 174.78, 174.36, 174.29,173.36, 169.00, 159.08 (q, J=284.4 Hz), 136.91, 133.88, 130.75, 125.20,123.30, 120.83, 116.67 (q, J=37.7 Hz), 102.29, 101.93, 96.83, 96.00,78.08, 76.57, 76.46, 76.30, 76.28, 76.01, 75.77, 73.36, 73.29, 70.65,70.55, 70.35, 70.22, 68.47, 67.35, 59.17, 53.76, 53.47, 52.67, 50.38,35.98, 32.35, 30.95, 28.15, 21.79. HRMS (ESI) m/z calcd forC₄₃H₅₉F₃N₇O₂₆ (M+H) 1146.3462, found 1146.3478.

Synthesis of Tetrasaccharide GlcNAc6N₃α1-4GlcAβ1-4GlcNH₂α1-4GlcAβ2AAF22-1. Compound GlcNAc6N₃α1-4GlcAβ1-4GlcNTFAα1-4GlcAβ2AAMe F21-1 (30 mg,0.029 mmol) was dissolved in 8 mL of H₂O. The pH of the solution wasadjusted to 10 by adding 1 N NaOH. After being vigorously stirred atr.t. for 1.5 hr, the reaction mixture was neutralized with DOWEX HCR-W2(H⁺) resin, filtered and concentrated. The residue was purified by flashcolumn chromatography (EtOAc:MeOH:H₂O=4:2:1, by volume) to obtain awhite solid GlcNAc6N₃α1-4GlcAβ1-4GlcNH₂α1-4GlcAβ2AA F22-1 in 81% yield.¹H NMR (600 MHz, D₂O) δ 8.12-8.13 (d, J=7.8 Hz, 1H), 7.85-7.87 (d, J=7.8Hz, 1H), 7.48-7.50 (t, J=7.2 Hz, 1H), 7.20-7.22 (t, J=7.8 Hz, 1H),5.59-5.60 (d, J=3.6 Hz, 1H), 5.41-5.40 (d, J=4.2 Hz, 1H), 4.45-4.47 (d,J=7.8 Hz, 1H), 4.27-4.28 (d, J=7.8 Hz, 1H), 3.88-3.94 (m, 2H), 3.79-3.88(m, 6H), 3.66-3.78 (m, 8H), 3.62-3.64 (m, 2H), 3.46-3.52 (m, 2H),3.34-3.37 (t, J=7.8 Hz, 1H), 3.19-3.33 (m, 3H), 2.73-2.75 (t, J=6.6 Hz,2H), 2.60-2.62 (t, J=6.6 Hz, 2H), 2.03 (s, 3H), 1.71-1.75 (m, 2H). ¹³CNMR (150 MHz, D₂O) δ 174.89, 174.87, 174.67, 174.47, 174.29, 172.74,137.25, 131.59, 130.48, 125.10, 124.00, 120.70, 102.27, 101.92, 96.84,95.32, 77.32, 76.22, 76.15, 76.10, 76.07, 75.79, 73.38, 73.03, 70.90,70.65, 70.34, 70.20, 68.24, 68.22, 67.30, 58.92, 53.82, 53.45, 50.38,35.90, 33.19, 31.28, 28.10, 21.77. HRMS (ESI) m/z calcd for C₄₀H₅₈N₇O₂₅(M+H) 1036.3482, found 1036.3497.

Synthesis of Tetrasaccharide GlcNAc6N₃α1-4GlcAβ1-4GlcNSα1-4GlcAβ2AAF22-2. Compound GlcNAc6N₃α1-4GlcAβ1-4GlcNH₂α1-4GlcAβ2AA F22-1 (20 mg,0.018 mmol) was dissolved in 10 mL of H₂O. The pH of the solution wasadjusted to 9.5 by adding 2 N NaOH (aq). Sulfur trioxide-pyridinecomplex (58 mg, 0.36 mmol) was added in three equal portions during 35minutes intervals at room temperature, and the pH was maintained at 9.5throughout the whole process using 2 N NaOH (aq). After being stirred atr.t. for 24 hr, the reaction mixture was neutralized with DOWEX HCR-W2(H⁺) resin, filtered, concentrated. The process has been repeated forthree times and purified using silica gel column (EtOAc:MeOH:H₂O=5:2:1,by volume) to obtain a light yellow solidGlcNAc6N₃α1-4GlcAβ1-4GlcNSα1-4GlcAβ2AA F22-2 in 70% yield. ¹H NMR (800MHz, D₂O) δ 8.13-8.14 (d, J=8.0 Hz, 1H), 7.87-7.88 (d, J=7.2 Hz, 1H),7.52-7.50 (t, J=8.0 Hz, 1H), 7.22-7.24 (t, J=7.2 Hz, 1H), 5.60-5.61 (d,J=4.0 Hz, 1H), 5.42-5.43 (d, J=3.2 Hz, 1H), 4.50-4.51 (d, J=8.0 Hz, 1H),4.34-4.35 (d, J=8.0 Hz, 1H), 3.86-3.92 (m, 3H), 3.80-3.83 (m, 4H),3.73-3.79 (m, 4H), 3.68-3.72 (m, 4H), 3.64-3.65 (d, J=3.2 Hz, 2H),3.56-3.53 (m, 1H), 3.48-3.50 (t, J=9.6 Hz, 1H), 3.36-3.39 (t, J=8.0 Hz,1H), 3.19-3.28 (m, 4H), 2.75-2.77 (t, J=7.2 Hz, 2H), 2.62-2.63 (t, J=7.2Hz, 2H), 2.04 (s, 3H), 1.75-1.77 (m, 1H). ¹³C NMR (200 MHz, D₂O) δ174.89, 174.51, 174.32, 174.30, 172.82, 172.78, 137.13, 131.55, 130.41,125.21, 124.02, 120.77, 102.11, 101.88, 96.88, 96.79, 77.70, 76.48,76.29, 76.15, 76.11, 75.77, 73.28, 72.51, 70.58, 70.30, 70.21, 70.15,69.38, 67.25, 59.16, 57.45, 53.43, 50.30, 35.89, 33.09, 31.19, 28.09,21.73. HRMS (ESI) m/z calcd for C₄₀H₅₈N₇O₂₈S (M+H) 1116.3051, found1116.3076.

Synthesis of Tetrasaccharide GlcNAc6NH₂α1-4GlcAβ1-4GlcNSα1-4GlcAβ2AAF22-3. Compound GlcNAc6N₃α1-4GlcAβ1-4GlcNSα1-4GlcAβ2AA F22-2 (17 mg,0.015 mmol) was dissolved in 10 mL H₂O/MeOH (1:1) and 20 mg of Pd/C wasadded. The mixture was shaken under H₂ gas (4 Bar) for 1 hr, filtered,and concentrated to produce F22-3 as a white solid in quantitativeyield. ¹H NMR (800 MHz, D₂O) δ 8.04-8.05 (d, J=8.0 Hz, 1H), 8.02-8.03(d, J=8.0 Hz, 1H), 7.64-7.67 (t, J=8.0 Hz, 1H), 7.32-7.34 (t, J=7.2 Hz,1H), 5.56-5.57 (d, J=4.0 Hz, 1H), 5.34-5.35 (d, J=4.0 Hz, 1H), 4.56-4.57(d, J=8.0 Hz, 1H), 4.34-4.35 (d, J=8.0 Hz, 1H), 3.92-3.95 (m, 3H),3.88-3.90 (dd, J=12.0, Hz, 2.4H), 3.76-4.84 (m, 4H), 3.68-3.75 (m, 4H),3.55-3.58 (m, 1H), 3.42-3.45 (dd, J=13.6, 3.2 Hz, 1H), 3.35-3.38 (m,2H), 3.30-3.32 (m, 2H), 3.19-3.27 (m, 4H), 3.12-3.15 (dd, J=12.8, 8.8Hz, 1H), 2.77-2.79 (t, J=6.4 Hz, 1H), 2.61-2.62 (t, J=6.4 Hz, 1H), 2.05(s, 3H), 1.75-1.78 (m, 2H). ¹³C NMR (200 MHz, D₂O) δ 174.77, 174.47,174.42, 173.26, 172.64, 170.76, 137.73, 133.92, 131.18, 127.27, 124.85,122.56, 102.18, 102.87, 97.64, 97.48, 77.66, 77.32, 76.56, 75.76, 75.73,75.70, 74.58, 73.42, 72.45, 71.70, 70.65, 70.08, 69.22, 68.16, 67.53,59.22, 57.60, 53.33, 46.57, 40.22, 35.88, 31.12, 28.13, 21.78. HRMS(ESI) m/z calcd for C₄₀H₆₀N₅O₂₈S (M+H) 1090.3146, found 1190.3171.

Synthesis of Tetrasaccharide GlcNAc6NSα1-4GlcAβ1-4GlcNSα1-4GlcAβ2AAF22-4. Compound GlcNAc6NH₂α1-4GlcAβ1-4GlcNSα1-4GlcAβ2AA F22-3 (14 mg,0.013 mmol) was dissolved in 5 mL of H₂O. The pH of the solution wasadjusted to 9.5 by adding 2 N NaOH. Sulfur trioxide-pyridine complex (30mg, 0.18 mmol) was added in three equal portions during 1 hr intervalsat rt. The pH was maintained at 9.5 throughout the whole process byadding 2 N NaOH. After being stirred at r.t. for overnight, the reactionmixture was neutralized with DOWEX HCR-W2 (H⁺) resin, filtered,concentrated. The process has been repeated for three times and purifiedby preparative HPLC using C18 column to give white solidGlcNAc6NSα1-4GlcAβ1-4GlcNSα1-4GlcAβ2AA F22-4 in 65% yield. ¹H NMR (800MHz, D₂O) δ 8.06-8.07 (d, J=8.0 Hz, 1H), 8.03-8.04 (d, J=8.0 Hz, 1H),7.66-7.68 (t, J=7.2 Hz, 1H), 7.33-7.34 (d, J=7.2 Hz, 1H), 5.54-5.55 (d,J=3.2 Hz, 1H), 5.35-5.36 (d, J=3.2 Hz, 1H), 4.61-4.62 (d, J=8.0 Hz, 1H),4.34-4.35 (d, J=8.0 Hz, 1H), 4.11-4.12 (d, J=9.6 Hz, 1H), 3.81-3.93 (m,4H), 3.73-3.81 (m, 5H), 3.64-3.71 (m, 5H), 3.55-3.58 (m, 1H), 3.50-3.53(t, J=9.6 Hz, 1H), 3.39-3.41 (t, J=8.0 Hz, 1H), 3.28-3.33 (m, 3H),3.21-3.27 (m, 3H), 2.77-2.79 (t, J=7.2 Hz, 2H), 2.61-2.62 (t, J=6.4 Hz,2H), 2.05 (s, 3H), 1.75-1.78 (m, 2H). ¹³C NMR (200 MHz, D₂O) δ 174.48,174.34, 173.25, 171.88, 171.81, 170.41, 137.88, 134.18, 131.28, 127.18,124.87, 122.59, 102.27, 102.04, 97.78, 97.46, 77.88, 76.83, 76.46,75.65, 75.49, 73.91, 73.88, 73.14, 72.41, 70.83, 70.69, 70.46, 70.13,69.14, 67.62, 59.21, 59.15, 57.67, 53.42, 43.29, 35.85, 32.71, 31.09,28.13, 21.81. HRMS (ESI) m/z calcd for C₄₀H₆₀N₅O₃₁S₂ (M+H) 1170.2714,found 1170.2730.

Alternative Route for Synthesizing Tetrasaccharide F22-4 from F21-1.

Compound GlcNAc6N₃α1-4GlcAβ1-4GlcNH₂α1-4GlcAβ2AAMe F21-1 (10 mg, 0.009mmol) was dissolved in 5 mL of H₂O/MeOH (1:1) and 5 mg of Pd/C wasadded. The mixture was shaken under H₂ gas (4 Bar) for 1 hr, filteredand concentrated to provide GlcNAc6NH₂α1-4GlcAβ1-4GlcNH₂α1-4GlcAβ2AA.The residue was dissolved in 5 mL of H₂O. The pH of the solution wasadjusted to 9.5 by adding 2 N NaOH. Sulfur trioxide-pyridine complex (15mg, 0.09 mmol) was added in three equal portions during 1 h intervals atrt. The pH was maintained at 9.5 throughout the whole process by adding2 N NaOH. After being stirred at r.t. for overnight, the reactionmixture was neutralized with DOWEX HCR-W2 (H⁺) resin, filtered, andconcentrated to give a mixture ofGlcNAc6NSα1-4GlcAβ1-4GlcNH₂α1-4GlcAβ2AA,GlcNAc6NH₂α1-4GlcAβ1-4GlcNSα1-4GlcAβ2AA (F22-3), andGlcNAc6NSα1-4GlcAβ1-4GlcNSα1-4GlcAβ2AA (F22-4) which can be separated byHPLC using a C18 column.

Synthesis of GlcNSα1-4GlcAβ1-4GlcNSα1-4GlcAβ2AA. The synthesis wasconducted as outlined in FIG. 14.

Trisaccharide GlcAβ1-4GlcNTFAα1-4GlcAβ2AAMe (Compound F14-1 or F20-2,FIG. 14) (11 mg, 1 eq.), GlcNTFA (1.5 eq.), ATP (1.8 eq.), and UTP (1.8eq.) were dissolved in water in a 15 mL centrifuge tube containing trisbuffer (100 mM, pH 7.0) and MgCl₂ (10 mM). After the addition ofNanK_ATCC55813 (2.5 mg), PmGlmU (3 mg), PmPpA (1.5 mg), and PmHS2 (2mg), water was added to bring the volume of the reaction mixture to 10mL. The reaction was carried out by incubating the solution in anisotherm incubator at 37° C. for 20 hr with gentle shaking. Productformation was monitored by TLC (EtOAc:MeOH:H₂O=4:2:1 by volume) withp-anisaldehyde sugar staining. The reaction was stopped by adding thesame volume of ice-cold ethanol and incubating at 4° C. for 30 min. Themixture was concentrated and passed through a BioGel P-2 gel filtrationcolumn to obtain the desired product. The tetrasaccharide was furtherpurified by silica gel column chromatography (EtOAc:MeOH:H₂O=5:2:1) toobtain a white solid of GlcNTFAα1-4GlcAβ1-4GlcNTFAα1-4GlcAβ2AAMe(Compound F14-2, FIG. 14). 11.8 mg, 84% yield. ¹H NMR (600 MHz, D₂O) δ8.04-8.03 (d, J=8.4 Hz, 1H), 7.87-7.85 (d, J=7.8 Hz, 1H), 7.72-7.69 (t,J=7.2 Hz, 1H), 7.41-7.38 (t, J=7.8 Hz, 1H), 5.54-5.53 (d, J=4.2 Hz, 1H),5.50-5.49 (d, J=3.6 Hz, 1H), 4.56-4.55 (d, J=7.8 Hz, 1H), 4.38-4.36 (d,J=7.8 Hz, 1H), 4.00-3.26 (m, 24H), 2.83-2.81 (t, J=6.9 Hz, 2H),2.68-2.66 (t, J=6.9 Hz, 2H), 1.82-1.80 (m, 2H).

GlcNTFAα1-4GlcAβ1-4GlcNTFAα1-4GlcAβ2AAMe (Compound F14-2, FIG. 14) (11mg) was dissolved in 7.5 mL solution of(MeOH:H₂O:triethylamine=1:1:0.5). The reaction was stirred overnight andmonitored until completion as indicated by TLC. The solution was thenrotovaped and re-dissolved in water and lyophilized to afford freeamines as a white foam. The free amine was then dissolved in 7 mL ofwater and the pH of the solution was adjusted to 9.5 by adding 2 N NaOH(aq). Sulfur trioxide-pyridine complex (60 mg, 0.37 mmol) was added inthree equal portions during 35 minutes intervals at room temperature,and the pH was maintained at 9.5 throughout the whole process using 2 NNaOH (aq). After being stirred at r.t. for 24 hr, the reaction mixturewas neutralized with DOWEX HCR-W2 (H⁺) resin, filtered, concentrated.The crude product was purified using silica gel column(EtOAc:MeOH:H₂O=5:3:2, v/v) to obtain a light yellow solidGlcNSα1-4GlcAβ1-4GlcNSα1-4GlcAβ2AA (tetrasaccharide F14-3, FIG. 14). MS(ESI) m/z calcd for C₃₈H₅₆N₄O₃₁S₂ (M−H) 1127.23, found 1127.23.C₃₈H₅₆N₄O₃₁S₂ (M/2−H) 563.11, found 563.11.

Results and Discussion

As shown in FIG. 16, four enzymes were used in one-pot to synthesizeGlcNAcα1-4GlcA disaccharide derivatives. The first enzyme was anN-acetylhexosamine 1-kinase cloned from Bifidobacterium infantis strainATCC15697 (NahK_ATCC15697). The second enzyme was anN-acetylglucosamine-1-phosphate uridylyltransferase that we cloned fromPasteurella multocida strain P-1059 (ATCC15742) (PmGlmU). The thirdenzyme was an inorganic pyrophosphatase that we cloned from Pasteurellamultocida strain P-1059 (PmPpA) for hydrolyzing the pyrophosphateby-product formed to drive the reaction towards the formation ofUDP-GlcNAc and derivatives.

The fourth enzyme is a heparosan synthase 2 cloned from Pasteurellamultocida strain P-1059 (PmHS2) for the formation of α1-4 linkage. PmHS2is a bifunctional enzyme which demonstrates α1-4GlcNAc and β1-4GlcAtransferase activity. It not only uses UDP-GlcNAc as donor, transferringGlcNAc to GlcA to form α1-4 linkage, but also transfers GlcA from donorUDP-GlcA to acceptor GlcNAc to form β1-4 linkage. Although PmHS2 hasbeen shown to be able to synthesize heparosan polysaccharides, its donorand acceptor specificity has not been investigated in detail.

Prior to applying the one-pot three-enzyme system shown in FIG. 16 tothe preparative-scale synthesis of the disaccharides, UDP-GlcNAc andderivatives F17-1-F17-12 were tested as donor substrates for PmHS2 insmall-scale reaction containing Tris-HCl buffer (100 mM, pH 7.5),GlcAβ2AAMe (10 mM), UDP-GlcNAc or a derivative (15 mM), MgCl₂ (10 mM),and PmHS2 (0.5 mg/mL). See FIG. 17. The reactions were carried out at37° C. for 12 hr and analyzed by thin layer chromatography (TLC).UDP-GlcNAc F17-1 and some of its C2-(UDP-GlcNTFA F17-2, UDP-GlcNGcF17-3, and UDP-GlcNAcN₃ F17-4), and C6-(UDP-GlcNAc6N₃ F17-8 andUDP-GlcNAc6NGc F17-9) derivatives are tolerable donor substrates forPmHS2. UDP-GlcNH₂ F17-5, UDP-GlcN₃ F17-6, UDP-GlcNS F17-7,UDP-GlcNAc6NH₂ F17-10, UDP-GlcNAc6NAcN₃ F17-11 and UDP-GlcNAc6S F17-12did not serve as donor substrates for PmHS2.

As shown in FIG. 18, preparative-scale transfer of monosaccharideGlcNAc, GlcNTFA and GlcNAc6N₃ to fluorescent labeled glucuronideGlcAβ2AAMe as an acceptor for PmHS2 successfully produced disaccharidesGlcNAcα1-4GlcAβ2AAMe F18-1, GlcNTFAα1-4GlcAβ2AAMe F18-2, andGlcNAc6N₃α1-4GlcAβ2AAMe F18-3 in 95%, 84%, and 89% yields, respectively.It was found that the N-TFA group removal was significant at pH 7.5.Nevertheless, the removal of N-TFA was not significant when the pH ofthe reaction mixture was changed from 7.5 to 6.5 and the reaction timewas shortened. Three additional disaccharides (supporting information)were also synthesized by PmHS2-catalyzed reaction using UDP-GlcNAcderivatives UDP-GlcNGc F17-3, UDP-GlcNAcN₃ F17-4, and UDP-GlcNAc6NGcF17-9, since the three sugar nucleotides were prepare form UDP-GlcNTFAF17-2 by the removal of TFA and acylation of amine with proper acylchloride. GlcNGcα1-4GlcAβ2AAMe F18-4, GlcNAcN₃α1-4GlcAβ2AAMe F18-5, andGlcNAc6NGcα1-4GlcAβ2AAMe F18-6 are prepared in 92%, 91%, and 74% yields,respectively. See FIG. 18.

Acceptor specificity of the β1-4GlcA transferase activity of PmHS2 wasalso explored in one-pot three-enzyme system, as shown in FIG. 19. Thefirst enzyme was a glucose-1-phosphate uridylyltransferase (GalU) whichcatalyzes the reversible conversion of Glc-1-P in the presence of UTP toproduce UDP-Glc and inorganic pyrophosphate. The second enzyme was aUDP-glucose dehydrogenase (Ugd) for oxidation of 6-OH in glucose residueof UDP-Glc to form the UDP-glucuronic acid (UDP-GlcA) in the presence ofits coenzyme NAD⁺. The third enzyme is PmHS2 transferring GlcA fromUDP-GlcA for the formation of β1-4 linkage. As shown in FIG. 20,trisaccharides GlcAβ1-4GlcNAcα1-4GlcAβ2AAMe F20-1,GlcAβ1-4GlcNAc6N₃α1-4GlcAβ2AAMe F20-3, GlcAβ1-4GlcNAcN₃α1-4GlcAβ2AAMeF20-5 were synthesis by small-scale reaction and analyzed by HPLC methodin 100%, 100% and 95% yields, respectively. The relative low yield (72%)for the formation of GlcAβ1-4GlcNTFAα1-4GlcAβ2AAMe F20-2 was due to theformation of byproduct GlcAβ1-4GlcNH₂α1-4GlcAβ2AAMe in which the TFAgroup was removed. Disaccharide F18-4 with N-glycolyl group in C2position of glucosamine residue acts as a good acceptor for PmHS2,leading to the formation of GlcAβ1-4GlcNGcα1-4GlcAβ2AAMe F20-4 in 75%yield, but the disaccharide F18-6 with N-glycolyl group in C6 positionof GlcNAc was converted to trisaccharideGlcAβ1-4GlcNAc6NGcα1-4GlcAβ2AAMe F20-6 only in 14% yield. Takentogether, these results indicate that the donor and acceptor substrateactivity of PmHS2 can tolerate a limited number of modifications on C-2and C-6 position of glucosamine residue.

Preparative-scale synthesis of trisaccharide F20-2 was also achieved.The removal of TFA group was significantly reduced when the pH of thereaction mixture was change for 7.5 to 7.0, and the yield increased to87% from 72%. Trisaccharide F20-2 was used as the starting material forthe synthesis of the tetrasaccharide F21-1 (FIG. 21). In the one-potfour-enzyme system, monosaccharide GlcNAc6N₃ was converted toGlcNAc6N₃-1-P by NanK, followed by the formation of UDP-GlcNAc6N₃ byPmGlmU, and transferred to trisaccharide F20-2 to obtainGlcNAc6N₃α1-4GlcAβ1-4GlcNTFAα1-4GlcAβ2AAMe F21-1 in 93% yield.

The N-TFA group as well as the N₃ group can be easily converted to afree amine, allowing sequential sulfation to generate a diverse array ofHS tetrasaccharides. As shown in FIG. 22, the N-TFA group at C2 ofinternal GlcNTFA residue of tetrasaccharide F21-1 was removed under mildbasic conditions to produce GlcNAc6N₃α1-4GlcAβ1-4GlcNH₂α1-4GlcAβ2AAF22-1 in 81% yield. As the removal of TFA group was accompanied bydemethylation in methyl carboxylic ester, tetrasaccharide F22-1 containa free carboxyl acid in 2AA motif instead of carboxylic ester intetrasaccharide F21-1. Conversion of F22-1 toGlcNAc6N₃α1-4GlcAβ1-4GlcNSα1-4GlcAβ2AA F22-2 (70%) needed a largerexcess of sulfating reagent (60 equiv.) and prolonged reaction time (3d). Catalytic hydrogenation of the azido group at the C6 of non-reducedend GlcNAc6N₃ generated GlcNAc6NH₂α1-4GlcAβ1-4GlcNSα1-4GlcAβAA F22-3 andfollowed by the sulfation to produceGlcNAc6NSα1-4GlcAβ1-4GlcNSα1-4GlcAβ2AA F22-4.

Example 9 Preparation of GlcA-TEG-PABA-Biotin

The synthesis was conducted as outlined in FIG. 15.

2-(2-(2-Tosylethoxy)ethoxy)ethanol. Compound F15-1 (51 grams) wasdissolved in 10 mL of water containing 2.2 grams of NaOH. The reactionmixture was cooled in an ice bath. To the reaction mixture tosylchloride (6.5 g, 34.1 mmol) in 80 mL of THF was added drop-wisely in 1hr period. The reaction was left in ice bath off for 2 hr. The reactionwas worked up with 150 mL of DCM and 150 mL of cold water. The organiclayer was collected and saved. The aqueous layer was extracted twicewith DCM (150 mL) and the organic layers were collected and washed twicewith water (200 mL). The organic portions were combined, dried withmagnesium sulfate, and rotovoped to provide crude2-(2-(2-tosylethoxy)ethoxy)ethanol (9.684 grams, 93% yield).

Compound F15-2. 2-(2-(2-Tosylethoxy)ethoxy)ethanol (9.684 g, 31.8 mmol)was dissolved in 25 mL of DMF. Sodium azide (10.34 g, 159.1 mmol) wasadded to the reaction solution. The reaction was left for 3 h at 80° C.The reaction mixture was worked up with EtOAc/water. The organic layerwas collected and dried over magnesium sulfate and purified by silicagel column (Hexane:EtOAc=2:1-0:1) to produce compound F15-2 (4.96 g, 89%yield).

Azidotriethylene Glycol-Boc. Compound F15-2 (2.334, 13.3 mmol) wasdissolved in 40 mL of DMF and cooled in ice bath. Sodium hydride 50%immersed in mineral oil (959 mg, 19.9 mmol) was added slowly. Thereaction was allowed to sit for 20 minutes followed by addition oft-butyl bromoacetate (3.93 mL, 39.9 mmol). The reaction was left forfour hours and extracted with ethylacetate and water. The organics weredried over magnesium sulfate and purified by silica gel column(Hexane:EtOAc=5:1-1:1) to afford azidotriethylene glycol-Boc (1.54 g;Yield: 40%; clear oil). NMR (600 MHz, CDCl₃) δ 4.01 (s, 2H), 3.71-3.65(m, 10H), 3.38-3.36 (t, J=4.5 Hz, 1H), 1.45 (s, 9H), ¹³C NMR (150 MHz,CDCl₃) δ 169.75, 81.61, 70.83, 70.79, 70.78, 70.76, 70.13, 69.15, 50.79,28.21.

Compound F15-3. Azidotriethylene glycol-Boc (1.19 g, 4.1 mmol) wasdissolved in 15 mL of ethyl acetate and of Pd/C catalyst (240 mg) wasadded under hydrogen gas in a double balloon. Reaction was stirred untilreaction was completed as monitored by TLC. The reaction mixture wasfiltered over celite and the filtrate was rotovaped to afford crudecompound F15-3. Yield: quant; clear oil.

Compound F15-5. D-Biotin F15-4 (538 mg, 2.2 mmol) was dissolved in 10 mLof hot DMF. The solution was allowed to cool to room temperature andHATU (838 mg, 2.2 mmol) was added and allowed to preactivate for 15minutes. To The reaction mixture diisopropylethylamine (0.968 mL, 2.4mmol) and para-amino benzoic acid (335 mg, 2.4 mmol) were added.Reaction was allowed to react for 24 hr in which then 70 mL ofdichloromethane was added to precipitate the product. The precipitatewas collected via suction filtration and washed three times with ethylacetate (50 mL) to attain NMR pure compound F15-5 (687 mg, Yield: 86%;white solid). ¹H NMR (600 MHz, DMSO) δ 10.19 (s, N—H), 7.88-7.86 (d,J=8.4 Hz, 2H), 7.71-7.69 (d, J=9.0 Hz, 2H), 6.46 (s, N—H), 6.38 (s,N—H), 4.32-4.29 (m, 1H), 4.15-4.12 (m, 1H), 3.13-3.10 (m, 1H), 2.83-2.80(dd, J=12.6 Hz, 5.4 Hz, 1H), 2.59-2.57 (d, J=12.6, 1H), 2.36-2.33 (t,J=7.5 Hz, 2H), 1.67-1.34 (m, 6H). ¹³C NMR (150 MHz, DMSO) δ 171.75,166.97, 162.77, 143.35, 130.38, 124.90, 118.26, 61.07, 59.22, 55.4039.94, 36.33, 28.23, 28.11, 24.99.

Compound F15-6. Compound F15-5 (1.116 g, 3.1 mmol) was dissolved in 15mL of DMF. carbonyldiimidazole (547 mg, 3.4 mmol) was added to reactionmixture and allowed to preactivate for 40 minutes followed by theaddition of compound F15-3 (977 mg, 3.7 mmol) in 5 mL of DMF. Thereaction was left at room temperature for 40 hours and was passedthrough silica gel column (DCM:MeOH:NH₄OH=9:1:0.1-1:1:0.1) to affordF15-6 (738 mg; Yield: 40%; yellow flakes). ¹H NMR (600 MHz, DMSO) δ10.13 (s, N—H), 8.40 (m, N—H), 7.89-7.77 (d, J=8.4 Hz, 2H), 7.65-7.64(d, J=9.0 Hz, 2H), 6.45 (s, N—H), 6.38 (s, N—H), 4.32-4.30 (m, 1H),4.15-4.13 (m, 1H), 3.95 (s, 2H) 3.53-3.50 (m, 10H), 3.40-3.38 (m, 2H),3.13-3.10 (m, 1H), 2.83-2.80 (dd, J=12.6 Hz, 4.8 Hz, 1H), 2.59-2.57 (d,J=12.6, 1H), 2.34-2.31 (t, J=7.2 Hz, 2H), 1.67-1.34 (m, 15H). ¹³C NMR(150 MHz, DMSO) δ 171.86, 169.57, 166.07, 163.03, 141.91, 128.64,128.06, 118.18, 80.89 70.0, 69.85, 69.83, 69.75, 69.1, 68.26, 61.28,59.42, 55.56, 40.00, 39.25, 36.43, 28.42, 28.25, 27.90 25.19.

Compound F15-7. Compound F15-6 (701 mg) was dissolved in 7 mL mixture ofDCM/TFA (2:1) and was left for 3 hr. The crude product was passedthrough silica gel column (DCM:MeOH:NH₄OH=7:3:0.1-0:1:0.1) to afford thefree acid (562 mg, 88% yield). The free acid (150 mg, 0.27 mmol) andN-hydroxy succamide (32 mg, 0.3 mmol) were dissolved in hot DMF. Thereaction mixture was cooled to room temperature andN,N′-dicyclohexylcarbodiimide (67 mg, 0.35 mmol) was added and left for18 h. the reaction mixture was filtrate over celite and the filtrate wasrotovaped and the triturated with diethyl ether and collected by suctionfiltration to afford crude compound F15-7 (172 mg; Yield: 98%; whitesolid).

Glucoronic acid-β-propylamine (200 mg, 0.79 mmol) (obtained by reductionfrom compound F13-7) and compound F15-7 (672 mg, 1.03 mmol) weredissolved in dry methanol (15 mL) and stirred overnight. The reactionmixture was rotpavoped and purified by silica gel column(DCM:MeOH:NH₄OH=7:3:0.1-0:1:0.1) to provide GlcA-TEG-PABA-biotin (F15-8)(192 mg; Yield: 31%; white foam). ¹H NMR (600 MHz, D₂O) δ 7.74-7.73 (d,J=7.8 Hz, 2H), 7.56-7.54 (d, J=9.0 Hz, 1H), 4.56-4.54 (m, 1H), 4.39-4.38(d, J=7.8, 1H), 4.36-4.34 (m, 1H), 3.92 (s, 2H) 3.91-3.88 (m, 1H),3.71-3.47 (m, 18H), 3.37-3.35 (t, J=6.3 Hz, 2H), 2.94-2.91 (dd, J=12.6Hz, 4.8 Hz, 1H), 2.74-2.72 (d, J=12.0, 1H), 2.39-2.36 (t, J=7.5 Hz, 2H),1.80-1.38 (m, 8H), ¹³C NMR (150 MHz, D₂O) δ 175.60, 175.21, 172.09,169.54, 165.15, 140.69, 129.21, 128.16, 120.29, 102.43, 102.02, 75.95,75.47, 72.90, 72.87, 71.76, 70.19, 69.36, 68.74, 67.50, 61.98, 60.15,55.29, 39.63, 39.45 36.29, 35.79, 28.37, 28.13, 27.96, 27.67, 24.93.

Example 10 Preparation of GlcAβ1-4GlcNAc Disaccharide Derivatives

PmHS2 acceptor substrate specificities using UDP-GlcA as a donorsubstrate and α- and β-linked GlcNAc derivatives as acceptors werestudied. Conversion to the disaccharide products was estimated by LCMSand TLC analysis as outlined in Table 9 below.

TABLE 9 Reaction conditions and yields for the formation of GlcA-GlcNAcdissacharide derivatives. Acceptor GlcNAcα2AA GlcNAcβMU GlcNAcαProN₃GlcNAcβProN₃ Conditions (5 mM) (5 mM) (10 mM) (10 mM) MES (0.1M pH 6.5)100 mM 100 mM 100 mM 100 mM MnCl₂ 10 mM 10 mM 10 mM 10 mM UDP-GlcA 6 mM6 mM 12 mM 12 mM PmHS2 0.55 μg 0.55 μg 0.55 μg 0.55 μg Total volume 10μL 10 μL 10 μL 10 μL Reaction time/Temp 19 h/37° C. 19 h/37° C. 19 h/37°C. 19 h/37° C. Product (% yield) 33% based on 57% based on 30% based onTLC 60% based on TLC HPLC (UV) HPLC (UV)

As shown in FIG. 31 and FIG. 32, TLC and LC-MS data indicated that bothα- and β-linked GlcNAc with different aglycons are suitable acceptorsubstrates for PmHS2. Therefore, both GlcA and GlcNAc can be used as thefirst sugar for oligosaccharide synthesis by the methods described inthis invention.

Example 11 Inhibition Assays of Monosaccharides, Disaccharides,Trisaccharides, and Tetrasaccharides

Materials. Recombinant human fibroblast growth factors FGF-1, FGF-2,FGF-4, and anti-human FGF-1, FGF-2, FGF-4 were purchased from PeproTechInc (Rocky Hill, N.J.). Heparin-biotin was from Sigma (St. Louis, Mo.).Low molecular weight heparin (LMWH) was bought from AMS Biotechnology(Lake Forest, Calif.). Alexa Fluor® 488 goat anti-rabbit IgG (H+L) wasfrom Invitrogen (Carlsbad, Calif.). 384-Well NeutrAvidin-coated platesfor the sialidase assays were from Fisher Biotech.

Methods. All assays were carried out in duplicate in 384-wellNeutrAvidin coated plates. Heparin-biotin (20 μL, 2 μM) was added toeach well and the plate was incubated at 4° C. for overnight. The platewas washed with 3 rounds of 1×PBS buffer containing 0.05% Tween-20 andblocked with 1% BSA (50 μL for each well) and incubated at r.t. for 30min. After the plate was washed three times with 1×PBS buffer containing0.05% Tween-20, each set of duplicate wells were added 20 μL of humanFGF-1, FGF-2, or FGF-4 (1 μM) with or without premixing with LMWH (˜22nM or 0.1 μM), monosaccharide, or oligosaccharides (100 μM or 1 mM) andthe plate was incubate at r.t. for 1 hr. After washing three times with1×PBS buffer containing 0.05% Tween-20, anti-human FGF-1, FGF-2 or FGF-4(20 μL) was added and the plate was incubated at r.t. for 1 hr. Afterthe plate was washed three times with 1×PBS buffer containing 0.05%Tween-20, Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (20 μL) was addedand the plate was incubated at r.t. for 1 hr. After washed three timeswith 1×PBS buffer containing 0.05% Tween-20 and once with water, thefluorescent signals of wells in the plate were measured using amicrotiter plate reader.

Results. LMWH was used as a control sample for testing the inhibitoryactivities of sixteen compounds including seven monosaccharides, twodisaccharides, two trisaccharides, and five tetrasaccharides (see FIG.24 for compound structures) against the binding of fibroblast growthfactors FGF-1, FGF-2, and FGF-4 to heparin-biotin immobilized onNeutrAvidin-coated plates. See Table 10 and FIG. 23.

TABLE 10 Percentage inhibition of compounds F24-1-F24-16 (1 mM) againstthe binding of human FGF-1, FGF-2, and FGF-4 to heparin-biotinimmobilized on NeutrAvidin- coated plates. FGF-1 (% FGF-2 (% FGF-4 (%Compounds Structures inhibition) inhibition) inhibition) F24-1 GlcA 6568 54 F24-11 GlcAβ1-4GlcNH₂α1-4GlcAβ2AA 76 — — F24-13GlcNAc6N₃α1-4GlcAβ1-4GlcNH₂α1-4GlcAβ2AA 62 — — F24-15GlcNAc6NH₂α1-4GlcAβ1-4GlcNSα1-4GlcAβ2AA — 68 43 F24-16GlcNAc6NSα1-4GlcAβ1-4GlcNSα1-4GlcAβ2AA 58 55 —

Example 12 Substrate Specificity of KfiA and PmHS2

Materials and Methods

PmHS2, KfiA, and related fusion proteins were expressed, purified, andcharacterized as described in Example 1. Reactions with UDP-sugars werecarried out in duplicate at 37° C. in MES (100 mM, pH 6.5) containingUDP-GlcNAc or one of its derivatives (1 mM), GlcAβ2AA (1 mM), MnCl₂ (10mM) and KfiA. (2.8 μg μl⁻¹) or PmHS2 (1.1 μg μl⁻¹) for 24 h. Thereaction mixtures were stopped by adding ice-cold 10% (v/v) acetonitrileto make 100-fold dilutions and the mixtures were analyzed by HPLC asdescribed for the pH profile.

Results

Substrate Specificity of the GlcNAcT Activities of MBP-KfiA-His₆ andHis₆-PmHS2. As shown in FIG. 33, the donor substrate specificities ofthe α1-4-N acetylglucosaminyltransferase (α1-4GlcNAcT) activities of therecombinant MBP-KfiA-His₆ and His₆-PmHS2 were investigated usingGlcAβ2AA as an acceptor and a library of twenty three UDP-sugarsincluding UDP-GlcNAc (33-1) and its C2′- (33-2-33-9) or C6′-modified(33-10-33-16) derivatives, UDP-glucose (UDP-Glc, 33-17),UDP-N-acetylgalactosamine (UDP-GalNAc, 33-18), UDP-galactose (UDP-Gal,33-19), UDP-N-acetylmannosamine (UDP-ManNAc, 33-20) and its derivativewith a C2′-N3 modification (33-22), UDP-mannose (33-22) and itsderivatives with a CT-fluorine modification (33-23). The reactions wereeasily analyzed by high performance liquid chromatography (HPLC)equipped with a fluorescence detector using an excitation wavelength of315 nm and an emission wavelength of 400 nm to determine the ratio ofthe fluorescent disaccharide product formed and the fluorescent GlcAβ2AAacceptor. The presence of each disaccharide product was confirmed byhigh resolution mass spectrometry (HRMS). The catalytic efficiency ofthe α1-4GlcNAcT activity of His₆-PmHS2 was higher than that ofMBP-KfiA-His₆ as a less molar amount of His₆-PmHS2 was needed to achievethe same yield under the same assay conditions. For the UDP-sugars andderivatives tested (33-1-33-23), the α1-4GlcNAcT activity of His₆-PmHS2exhibited a much better tolerance towards substrate modifications thanMBP-KfiA-His₆. For example, UDP-GlcNAc (33-1) and its C2′- (33-2-33-4)and C6′- (33-10) derivatives that can be used by MBP-KfiA-His₆ were alsosuitable donor substrates for His₆-PmHS2. Noticeably, UDP-GlcNAc6N3(33-10) is a much better substrate for His₆-PmHS2 than MBP-KfiA-His₆.Some UDP-sugars including UDP-GlcNAcPh (33-5, a C2′-derivative ofUDP-GlcNAc), several C6′-derivatives of UDP-GlcNAc such as UDPGlcNAc6NH₂(33-11), UDP-GlcNAc6NGc (33-12), and UDP-GlcNAc6NAcN₃ (33-13), as wellas UDP-Glc (33-17) and UDP-GalNAc (33-18) are tolerable donor substratesfor His₆-PmHS2 but not for MBPKfiA-His₆.

UDP-GlcNAc derivatives with a bulky N-diphenylacetyl group at C2′ (33-6)or C6′ (33-15) or with a C6′-N-phenyl acetyl substitution (33-14) werenot suitable donor substrates for either MBP-KfiAHis₆ or the α1-4GlcNAcTactivity of His₆-PmHS2. It seems that the N-acyl groups at CT ofUDP-GlcNAc derivatives are quite important for recognition by His₆-PmHS2as donor substrates. For example, UDP-GlcN₃ (33-7) with a C2′-azidogroup substitution and UDP-GlcNH₂ (33-8) with a C2′-amino groupsubstitution were not acceptable donor substrates by MBP-KfiA-His₆ orthe al-4GlcNAcT activity of His₆-PmHS2. In addition, N-sulfation at C2′(33-9) or O-sulfation at C6′ (33-16) also block the activities of bothMBP-KfiA-His₆ and His₆-PmHS2. UDP-Gal (33-19), UDPManNAc (33-20),UDP-mannose (33-22) and their derivatives (33-21 and 33-23) were nottolerable substrate for either enzymes.

Example 13 Preparation of UDP-Uronic Acid Compounds Using a One-PotMulti-Enzyme System

Materials. The cDNA library of Arabidopsis thaliana was purchased fromAMS Biotechnology (Lake Forest, Calif., USA). Restriction enzymesincluding NdeI and BamHI were purchased from New England BioLabs(Beverly, Mass., USA). Vector pET22b+ was purchased from Novagen (EMDBiosciences Inc., Madison, Wis., USA). Herculase-enhanced DNA polymerasewas purchased from Stratagene (La Jolla, Calif., USA). T4 DNA ligase and1 kb DNA ladder were from Promega (Madison, Wis., USA).Ni²⁺-nitrilotriacetic acid (NTA) agarose, QIAprep spin miniprep kit andQIAquick gel extraction kit were purchased from Qiagen (Valencia,Calif., USA). Bicinchoninic acid (BCA) protein assay kit was from PierceBiotechnology, Inc. (Rockford, Ill., USA). Escherichia coli DH5(electrocompetent cells and BL21 (DE3) chemically competent cells werepurchased from Invitrogen (Carlsbad, Calif., USA).

Galactose (Gal), N-Acetylgalactosamine (GalNAc), N-Acetylglucosamine(GlcNAc), Mannose (Man), N-Acetylmannosamine (ManNAc), Xylose (Xyl) werepurchased from Sigma Aldrich (Saint Louis, Mo., USA). GalNAcα/βProN₃were chemically synthesized in the group.

Cloning. To clone full length Arabidopsis thaliana glucuronokinase (EC2.7.1.43) (AtG1 cAK) (encoded by gene GLCAK1, DNA GenBank accessionnumber: NM_(—)111030, locus tag: AT3G01640; protein GenBank accessionnumber: NP_(—)566144) into pET22b+, forward primer used was 5′ACGCGTCGACATGGATCCGAATTCCACGG 3′ (SalI restriction site is bold andunderlined) and reverse primer was 5′ CCGCTCGAGTAAGGTCTGAATGTCAGAATCATTC3′ (Xhol restriction site is bold and underlined). Polymerase chainreaction (PCR) were performed in 50 μL total volume containing200 ng ofcDNA library, 0.2 μM of forward and reverse primers, 5μL of 5 XHerculase II buffer, dNTP mixture (0.2 mM), and 5 U of Herculase II DNApolymerase. The reaction was performed at an annealing temperature of 58° C. for 32 cycles. The PCR products were digested with Sall and Xhol,purified, and ligated at 16 ° C. overnight with pET22b+vectorpredigested with SalI and Xhol. The ligated product was transformed intoelectrocompetent E. coli DH5αcells. Selected clones were grown forminipreps and positive clones were verified by restriction mapping andDNA sequencing performed by Davis Sequencing Facility.

Protein expression and purification. The plasmid containing GLCAK1 wastransformed into E. coli BL21 (DE3) chemically competent cells forprotein expression. E. coli cells harboring the pET22b-AtGlcAK plasmidwere cultured in LB medium (10 g/L tryptone, 5 g/L yeast extract, and 10g/L NaC1) with ampicillin (100μg /mL) at 37 ° C. with rigorous shakingat 250 rpm in a C25KC incubator shaker (New Brunswick Scientific,Edison, N.J.) until the OD_(600 nm) of the culture reached0.8-1.0.Overexpression of the targeted proteins was achieved by adding0.15 mM of isopropyl-1-thio-β -D-galactopyranoside (IPTG) followed byincubation at 18 ° C. for 20 h with rigorous shaking at 250 rpm.

His₆-tagged (SEQ ID NO:22) protein was purified from cell lysate usingNi²⁻-NTA affinity column. To obtain cell lysate, cells were harvested bycentrifugation at 4,000 rpm (Sorvall) at 4 ° C. for 2 h. The cell pelletwas resuspended in lysis buffer (pH 8.0, 100 mM Tris-HCI containing 0.1%Triton X-100). Lysozyme (100μg/mL) and DNase I (5 μg/mL) were added tothe cell suspension. The mixture was incubated at 37 ° C. for 1 h withvigorous shaking (200rpm). Cell lysate was obtained as the supernatantby centrifugation at 12,000 rpm (Sorvall) at 4° C. for 20 min.Purification was performed by loading the supernatant onto a Ni²⁺-NTAcolumn pre-equilibrated with 10 column volumes of binding buffer (40 mMimidazole, 0.5 M NaCl, 50 mM Tris-HC1, pH 7.5). The column was washedwith 10 column volumes of binding buffer and 10 column volumes ofwashing buffer (40 mM imidazole, 0.5 M NaCl, 50 mM Tris-HC1, pH 7.5).Protein of interest was eluted with Tris-HC1 (pH 7.5, 50 mM) containingimidazole (200 mM) and NaC1 (0.5 M). The fractions containing thepurified enzyme were collected and dialyzed against Tris- HC1 buffer (pH7.5, 25 mM) containing 10% glycerol and 0.25 M NaCl. Dialyzed proteinswere stored at 4 ° C. Alternatively, fractions containing purifiedenzyme were dialyzed against Tris-HCl buffer (pH 7.5, 25 mM) and freezedried.

pH Profile. Typical enzymatic assays for pH profile studies wereperformed in a total volume of 20 μL containing GlcA (10 mM), ATP (10mM), MgCl₂ (20 mM) and AtGlcAK (412 ng) in various buffers (200 mM) withpH varying from 5.0 to 10.0. All reactions were allowed to proceed for15 min at 37° C. The reaction mixture was quenched by boiling water bathfor 1 min followed by adding 20 μL of ice cold 95% (v/v) ethanol. Thesamples were then centrifuged at 13,000 rpm for 2 min, and kept on iceuntil analyzed by a Beckman Coulter P/ACE MDQ Capillary Electrophoresis(CE) system equipped with a UV detector. A 50 cm capillary tubing (75 μmI.D., Beckman Coulter) was used. Assays were run at 25 kV with 25 mMsodium borate buffer (pH 9.8) for 22 min. Percent conversions werecalculated from the ratios of ATP and ADP, which were determined by UVabsorbance at 254 nm. All assays were carried out in duplicate, andstandard deviations were used to represent errors.

Effects of Metal Ions and EDTA. EDTA (5 mM), 20 mM of various divalentmetal salts (CaCl₂, CoCl₂, CuSO₄, MnCl₂, ZnCl₂) were mixed in a MOPSbuffer (pH 7.5, 100 mM) to analyze their effects on the kinase activityof AtGlcAK (412 ng) in 20 μL total volume containing 10 mM of ATP andGlcA. Reaction without EDTA or metal ions was used as a control.Reactions were quenched and assayed using the same method as those forpH profile. All reactions were performed in duplicate, and standarddeviations were used to represent errors.

Substrate Specificity Assays. Substrate specificity assays wereperformed under two conditions: low enzyme concentration assays areperformed in 20 μL reaction mixture containing 10 mM ATP, 10 mM sugarsubstrate, 20 mM MgCl₂, 100 mM MOPS pH 7.5 and 412 ng AtGlcAK at 37° C.for 15 min, while high enzyme concentration assays are performed in 20μL reaction mixture containing 10 mM ATP, 10 mM sugar substrate, 20 mMMgCl₂, 100 mM MOPS pH 7.5 and 2.1 μg AtGlcAK at 37 t for 60 min. Thereactions were quenched and assayed with the same method as those for pHprofile. All reactions were performed in duplicates.

Kinetics Assays. Kinetic parameters of GlcA were assayed in duplicate inreaction mixture of 20 μL, containing 100 mM MES buffer (pH 7.5), 20 mMof MgCl₂, 10 mM of ATP, different concentration of GlcA (0.5, 1.0, 2.0,and 5.0 mM, 10 mM and 20 mM), and AtGlcAK (206 ng). All reactions wereallowed to proceed at 37° C. for 15 min. Reaction with no GlcA was usedas negative control. The apparent kinetic parameters were obtained byfitting the experimental data (average values of duplicate assays) intothe Michaelis-Menten equation using Grafit 5.0.

Kinetic parameters of ATP were assayed in duplicate in reaction mixtureof 20 μL containing 100 mM MES buffer (pH 7.5), 20 mM of MgCl₂, 10 mM ofGlcA, different concentration of ATP (0.5, 1.0, 2.0, and 5.0 mM, 10 mMand 20 mM), and AtGlcAK (206 ng). All reactions were allowed to proceedat 37° C. for 15 min. Reaction with no GlcA was used as negativecontrol. The apparent kinetic parameters were obtained by fitting theexperimental data (average values of duplicate assays) into theMichaelis-Menten equation using Grafit 5.0.

All assays were performed in a Beckman Coulter P/ACE MDQ CapillaryElectrophoresis System (Fullerton, Calif., USA) equipped with a UVdetector. Reactions were stopped by adding 25 μL of ice-cold ethanol,centrifuged at 13,000 rpm for 2 min, and kept on ice until aliquots of154 were transferred into micro sample vials and subjected to CEanalysis. ATP samples with higher concentrations are further dilutedbefore injection. An eCAP Capillary Tubing (50 cm effective length, 75μm I.D., 375 μm O.D.) from Beckman Coulter was used to separate ATP andADP in a 25 mM sodium borate buffer (pH 9.8) under 25 kV voltages.Sample injections were achieved by pressurizing sample vial to 0.5 psifor 6 sec. Separations were achieved within 22 min and percentconversions were calculated from the ratios of sugar nucleotides andnucleotide triphosphates, which were determined by UV absorbance at 254nm.

LC-MS Analysis of AtGlcAK Activity. The product formation was monitoredby LC-MS using a LC-2010AXL High Performance Liquid Chromatography(HPLC) system linked with a LCMS-2020 mass spectrometer (ShimadzuScientific instrument Inc., Columbia, Md.). Liquid nitrogen was used asnitrogen gas source. A Shimadzu C18 column (5 μm particle size, 4.6mm×50 mm) was used to clean up the reaction. Mobile phase consists of30% acetonitrile (ACN) in water. The flow rate was set at 0.8 ml/min.Each run was set for 6 minutes and mass spectrometer was set to scan therange from 190 to 600 Da per second.

Results

Cloning, expression and purification of AtG1cAK. Full length Arabidopsisthaliana glucuronokinase (EC 2.7.1.43) (AtG1cAK) was amplified from thecDNA library of Arabidopsis thaliana, cloned in pET15b vector, andexpressed as an N-His₆-tagged (SEQ ID NO:22) fusion protein. Positiveclones were verified by restriction mapping and DNA sequencing performedby Davis Sequencing Facility. The DNA sequence of the insert matched tothe reported G1cAK1gene.

N-terminal His₆-tagged (SEQ ID NO:22) protein was overexpressed in E.coli BL21(DE3) using Luria-Bertani (LB) media. The recombinant proteinwas then purified from cell lysate using Ni²⁺-NTA affinity column.According to SDS-PAGE analysis, the recombinant protein of around 42 kDa(calculated molecular mass 42.2 kDa) can be obtained effectively in highpurity. The fractions containing the purified enzyme were collected anddialyzed against Tris-HCl buffer (pH 7.5, 20 mM) containing 30%glycerol. Dialyzed proteins were stored at −20° C. Alternatively,fractions containing purified enzyme were dialyzed against Tris-HC1buffer (pH 7.5, 20mM) and lyophilized. On average, 65 mg of purifiedprotein was obtained from 1 liter of cell culture.

pH Profile. A pH profile study of AtGlcAK was performed on a capillaryelectrophoresis (CE) system using Glucuronic acid (GlcA) and ATP assubstrate. The reaction mixture also contains MgCl₂ (20 mM). UVabsorbance at 254 nm was used for quantification. Percentage conversionsof ATP to ADP were used to represent the progress of the reaction.AtGlcAK has a relatively narrow pH range (FIG. 34), with an optimal pHof 7.5. The activity dropped fast below 7.0 or above 8.0. No activitydetectable below pH 5.0 or above 10.0.

EDTA and Metal Effects. Metal dependence of AtGlcAK was assayedfollowing a similar assay method (FIG. 35). With the presence of EDTA,AtGlcAK shows no kinase activity towards GlcA. Divalent metal (Mg²⁺,Mn²⁺, Ca²⁺ or Co²⁺) is required for its activity. However, Cu²⁺ and Zn²⁺do not fit in the catalytic site very well.

Substrate Specificity of AtGlcAK.

Substrate specificity of AtGlcAK was assayed using a capillaryelectrophoresis (CE) system, using ATP and a variety of monosaccharides(Table 11). It is very obvious that the carboxylic acid at C6 of thesugar substrate is critical for binding to the AtGlcAK. Only thoseacidic monosaccharides can be phosphorylated by AtGlcAK with detectableyields.

TABLE 11 Substrate specificity of AtGlcAK Percentage conversion of ATP(%) Low enzyme Conc. High enzyme Conc. (412 ng in 20 μL, 15 min, (2.1 μgin 20 μL, 60 min, Substrate 37° C.) 37° C.) GlcA 61.5 N.A. GalA  6.2  15.3 IdoA N.D. N.D. Glc N.D. <5 Gal N.D. N.D. Man N.D. N.D. Xyl N.D.<5 Ara N.D. N.D. N.A.: Not Assayed; N.D. Not Detected.

Kinetics Assays of AtGlcAK. Kinetic studies for the glucuronokinaseactivity of AtGlcAK with GlcA have been performed (Table 12). The enzymeamount was adjusted so that with 10 mM of ATP and GlcA, at 15 min thereaction rate was still in its linear range and the ATP conversion wasrelatively high. The reaction rate started to slow down after 20 min(FIG. 36).

TABLE 12 Kinetic parameters of AtGlcAK k_(cat) (s⁻¹) K_(m) (mM)k_(cat)/K_(m) (mM⁻¹s⁻¹) ATP 26.8 1.1 24.4 GlcA 25.4 1.3 19.5

LC-MS Analysis of AtGlcAK Activity. The AtGlcAK kinase activity wasconfirmed by LC-MS under negative mode. The peak of 273.05 represents[M−H]−, whereas the peak of 295.05 represents [M+Na−2H]− of the product.The reaction did not go to completion with a 1:1 ratio of ATP:GlcA.Complete conversion can be achieved with an excess amount of ATP.

Preparative Synthesis of Oligosaccharides Using AtGlcAK. The GlcA-1-Pgenerated from AtGlcAK was used by a UDP-sugar pyrophosphorylase (BLUSP)in a one-pot multiple-enzyme to form UDP-GlcA, which is then used assubstrate by a chondroitin synthase from Pasteurella multocida (PmCS), ahyaluronan synthase from Pasteurella multocida (PmHAS), or a heparosansynthase from Pasteurella multocida (PmHS2). PmCS is a polymerase whichcatalyzes the alternating addition of GlcAβ1-3 and GalNAcβ1-4 onto anoligosaccharide chain. GlcAβ1-3GalNAcβProN₃, a disaccharide derivative,was generated by Pasteurella multocida chondroitin sulfate(PmCS)-catalyzed reaction using GalNAcβProN₃ as an acceptor using aone-pot four-enzyme system containing AtGlcAK, BLUSP, Pasteurellamultocida inorganic phosphatase (PmPpA), and PmCS. PmHAS is a polymerasewhich catalyzes the alternating addition of GlcAβ1-3 and GlcNAcβ1-4 ontoan oligosaccharide chain. Similar to that described for thePmCS-catalyzed reaction, GlcAβ1-3GlcNAcβProN₃, a disaccharidederivative, was generated by Pasteurella multocida hyaluronan synthase(PmHAS)-catalyzed reaction using GlcNAcβProN₃ as an acceptor using aone-pot four-enzyme system containing AtGlcAK, BLUSP, Pasteurellamultocida inorganic phosphatase (PmPpA), and PmHAS. Similarly,GlcAβ1-4GlcNAcα1-4GlcAβProN₃ trisaccharide was produced by a one-potfour-enzyme system containing AtGlcAK, BLUSP, PmPpA, and theβ1-4-glucuronyltransferase (β1-4-GlcAT) activity of Pasteurellamultocida heparosan synthase 2 (PmHS2)-catalyzed reaction usingGlcNAcα1-4GlcAβProN₃ as an acceptor. These demonstrated the importantapplication of AtGlcAK in one-pot multienzyme chemoenzymatic syntheticschemes for synthesizing GlcA-containing compounds and possibly forother uronic acid-containing structures.

Example 14 Synthesis and Biological Activity of Novel DisialylHexasaccharides

As discussed above, HMOs have the potential for important therapeuticuses. Most of the HMO-related studies reported so far, however, usedmixtures of HMOs and thus the key active components of HMOs are notclear. Also except for a few, the functions of individual HMOs are notwell understood. This is mainly due to the lack of access to pureoligosaccharides in amount large enough for research, preclinical, andclinical studies. The present invention provides efficient enzymaticmethods for obtaining biologically important HMOs and derivatives.

DSLNT contains two sialic acid residues: one is linked to the terminalgalactose (Gal) residue via an α2-3-sialyl linkage; the other is linkedto the internal N-acetylglucosamine (GlcNAc) residue via an α2-6-sialyllinkage. Despite recent advances in the development of chemical methodsfor synthesizing sialic acid-containing structures, sialosides remain tobe challenging targets for chemical synthesis due to the intrinsicstructural feature of sialic acids (e.g. steric hindered anomeric carbonwith an electron-withdrawing carboxyl group in the sialic acid whichlowers the glycosylation efficiency and the lack of a neighbouringparticipating group that disallow the control of the stereospecificityin the formation of sialyl bonds). Therefore, chemical synthesis ofDSLNT in a free oligosaccharide oligosaccharide form has so far not beenreported although a glycosyl ceramide containing the same glycan portionwas reported by the Kiso group. Furthermore, despite the identificationof the activity of an α2-6-sialyltransferase that catalyzes theformation of the α2-6-linked sialic acid on the internal GlcNAc residuein DSLNT (e.g. in the livers of various animals and human as well as inhuman placenta, bovine mammary gland, human milk, and human mammarytumor although at a lower level), the gene for the enzyme has yet to bedetermined. Therefore, it is currently unfeasible to obtain the desiredα2-6-sialyltransferase in large amount via cloning to allow enzymaticsynthesis of DSLNT in large scale.

One feasible strategy is to identify compounds that have similar orbetter effects than DSLNT in treating NEC but can be easily obtainedsynthetically. Here, we report a novel synthetic α2-6-linkeddisialyllacto-N-neotetraose (DSLNnT) obtained by sequential one-potmultienzyme (OPME) reactions which shows potent effect in preventing NECin neonatal rats. The compound is readily available and can be producedin large amount for potential therapeutic application in treating NEC inpreterm infants.

Materials and Methods

General Methods. Chemicals were purchased and used without furtherpurification. ¹H NMR (800 MHz) and ¹³C NMR (200 MHz) spectra wererecorded on a Bruker Avance-800 NMR spectrometer. High resolutionelectrospray ionization (ESI) mass spectra were obtained using ThermoElectron LTQ-Orbitrap Hybrid MS at the Mass Spectrometry Facility in theUniversity of California, Davis. Silica gel 60 Å (200-425 mesh, FisherChemical) was used for flash column chromatography. Thin-layerchromatography (TLC) was performed on silica gel plates usinganisaldehyde sugar stain or 5% sulfuric acid in ethanol stain fordetection. Gel filtration chromatography was performed with a column(100 cm×2.5 cm) packed with Bio-Gel P-2 Fine resins.

One-Pot Four-Enzyme Preparative-Scale Synthesis of Lc₃ TrisaccharideGlcNAcβ1-3Galβ1-4Glc. To prepare the trisaccharide, a reaction mixturein Tris-HCl buffer (100 mM, pH 8.0) in a total volume of 65 mLcontaining lactose (0.90 g, 2.63 mmol), N-acetylglucosamine (GlcNAc,0.756 g, 3.42 mmol), adenosine 5′-triphosphate (ATP, 1.88 g, 3.42 mmol),uridine 5′-triphosphate (UTP, 1.99 g, 3.42 mmol), MgCl₂ (20 mM). NahK(19.0 mg), PmGlmU (8.0 mg), NmLgtA (6.0 mg), and PmPpA (4.0 mg) wasincubated in a shaker at 37° C. for 48 hrs. The product formation wasmonitored by TLC (EtOAc:MeOH:H₂O:HOAc=4:2:1:0.2 by volume and detectedby p-anisaldehyde sugar stain) and mass spectrometry. To stop thereaction, the reaction mixture was added with same volume (65 mL) ofethanol and incubated at 4° C. for 30 min. After centrifugation, thesupernatant was concentrated and passed through a Bio Gel P-2 gelfiltration column (water was used as an eluent). The fractionscontaining the product were collected, concentrated, and furtherpurified by silica gel column (EtOAc:MeOH:H₂O=5:2:1 by volume) toprovide Lc₃ trisaccharide GlcNAcβ1-3Galβ1-4Glc (1.36 g, 95%). ¹H NMR(800 MHz, D₂O) δ 5.19 (d, J=4.0 Hz, 0.4H), 4.66 (d, J=8.0 Hz, 0.4H),4.65 (d, J=8.0 Hz, 0.6H), 4.64 (d, J=8.0 Hz, 0.6H), 4.41 (d, J=8.0 Hz,1H), 4.12 (d, J=3.2 Hz, 1H), 3.93-3.24 (m, 17H), 2.01 (s, 3H). ¹³C NMR(200 MHz, D₂O) β-isomer: δ 174.87, 102.84, 102.75, 95.66, 81.87, 78.21,75.57, 74.80, 74.71, 74.20, 73.71, 73.49, 70.03, 69.92, 68.26, 60.88,60.41, 60.01, 56.58, 22.09. HRMS (ESI) m/z calculated for C₂₀H₃₆NO₁₆(M+H) 546.2034, found 546.2026.

One-Pot Four-Enzyme Preparative-Scale Synthesis of LNnTGalβ1-4GlcNAcβ1-3Galβ1-4Glc. To prepare LNnT, a reaction mixture inTris-HCl buffer (100 mM, pH 8.0) in a total volume of 80 mL containingLc₃ trisaccharide (1.0 g, 1.83 mmol), galactose (0.43 g, 2.38 mmol), ATP(1.40 g, 2.38 mmol), UTP (1.58 g, 2.38 mmol), MgCl₂ (20 mM), EcGalK(20.0 mg), BLUSP (20.0 mg), NmLgtB (15.0 mg), and PmPpA (20 mg) wasincubated in a shaker at 37° C. for 30 hrs. The product formation wasmonitored by TLC (n-PrOH:H₂O:NH₄OH=5:2:1 by volume and detected byp-anisaldehyde sugar stain) and mass spectrometry. When an optimal yieldwas achieved, the reaction mixture was added with the same volume (80mL) of ethanol and incubated at 4° C. for 30 min. The precipitates wereremoved by centrifugation and the supernatant was concentrated andpurified by a Bio-Gel P-2 gel column (water was used as an eluent).Further purification was achieved by silica gel chromatography(EtOAc:MeOH:H₂O=5:3:1.5 by volume) to produce LNnTGalβ1-4GlcNAcβ1-3Galβ1-4Glc (1.19 g, 92%). ¹H NMR (800 MHz, D₂O) δ 5.17(d, J=4.0 Hz, 0.4H), 4.66 (d, J=8.0 Hz, 0.4H), 4.65 (d, J=8.0 Hz, 0.6H),4.61 (d, J=8.0 Hz, 0.6H), 4.43 (d, J=7.2 Hz, 1H), 4.38 (d, J=8.0 Hz,1H), 4.11 (d, J=3.2 Hz, 1H), 3.91-3.87 (m, 2H), 3.84-3.22 (m, 21H), 1.98(s, 3H). ¹³C NMR (200 MHz, D₂O) β-isomer: δ 174.83, 102.79, 102.76,102.73, 95.61, 81.82, 78.21, 78.11, 75.52 (2C), 74.76, 74.66, 74.22,73.65, 73.42 (2C), 70.99, 69.88, 68.24, 68.22, 60.85, 60.84, 60.34,59.93, 56.52, 22.03. HRMS (ESI) m/z calculated for C₂₆H₄₅NO₂₁Na (M+Na)730.2382, found 730.2379.

One-Pot Two-Enzyme Preparative-Scale Synthesis of Hexasaccharide DSLNnTNeu5Acα2-6Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc. To prepare DSLNnT, areaction mixture in Tris-HCl buffer (100 mM, pH 8.5) in a total volumeof 10 mL containing LNnT (131 mg, 0.19 mmol), Neu5Ac (143 mg, 0.46mmol), CTP (260 mg, 0.46 mmol), MgCl₂ (20 mM), NmCSS (3.0 mg), andPda-2,6ST (2.0 mg) was incubated in a shaker at 37° C. for 36 hrs. Thereaction was monitored by TLC (n-PrOH:H₂O:NH₄OH=4:2:1 by volume anddetected by p-anisaldehyde sugar stain) and mass spectrometry. When anoptimal yield was achieved, the reaction mixture was added with the samevolume (10 mL) of ethanol and incubated at 4° C. for 30 min. Theprecipitates were removed by centrifugation and the supernatant wasconcentrated and purified by a Bio Gel P-2 gel column (water was used asan eluent). Further purification was achieved by silica gelchromatography (EtOAc:MeOH:H₂O=4:3:2 by volume) to produceNeu5Acα2-6Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc hexasaccharide (236mg, 99%). ¹H NMR (800 MHz, D₂O) δ 5.20 (d, J=4.0 Hz, 0.5H), 4.69 (d,J=8.0 Hz, 1H), 4.65 (d, J=8.0 Hz, 0.5H), 4.42 (d, J=8.0 Hz, 1H), 4.41(d, J=8.0 Hz, 1H), 4.17 (d, J=2.4 Hz, 1H), 3.99-3.28 (m, 37H), 2.68 (dd,J=4.8 and 12.8 Hz, 1H), 2.64 (dd, J=4.8 and 12.8 Hz, 1H), 2.03 (s, 3H),2.01 (s, 6H), 1.73-1.69 (m, 2H). ¹³C NMR (200 MHz, D₂O) β-isomer: δ175.01 (3C), 173.66, 173.59, 103.65, 103.35, 102.74, 100.38, 100.23,95.70, 82.29, 80.69, 79.76, 74.76, 74.72, 74.36, 73.80, 73.77, 73.37,72.64 (2C), 72.62, 72.50, 71.86, 71.81, 70.83, 69.76, 68.50 (2C), 68.47(3C), 68.32, 63.37, 63.23, 62.53 (2C), 60.23, 60.05, 54.83 (2C), 51.79,51.69, 40.17 (2C), 22.38, 21.15, 21.12. HRMS (ESI) m/z calculated forC₄₈H₇₈N₃O₃₇ (M−H) 1288.4314, found 1288.4305.

One-Pot Two-Enzyme Preparative-Scale Synthesis of Pentasaccharide3′″-sLNnT Neu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc. To prepare thepentasaccharide, a reaction mixture in Tris-HCl buffer (100 mM, pH 8.5)in a total volume of 8 mL containing LNnT (100 mg, 0.14 mmol), Neu5Ac(65 mg, 0.21 mmol), CTP (119 mg, 0.21 mmol), MgCl₂ (20 mM), NmCSS (2.0mg), and PmST1 M144D (1.5 mg) was incubated in a shaker at 37° C. for 48hrs. The reaction was monitored by TLC (n-PrOH:H₂O:NH₄OH=4:2:1 by volumeand detected by p-anisaldehyde sugar stain) and mass spectrometry. Whenan optimal yield was achieved, the reaction mixture was added with thesame volume (8 mL) of ethanol and incubated at 4° C. for 30 min. Theprecipitates were removed by centrifugation and the supernatant wasconcentrated and purified by a Bio-Gel P-2 gel column (water was used asan eluent). Further purification was achieved by silica gelchromatography (EtOAc:MeOH:H₂O=5:3:2 by volume) to produceNeu5Acα2-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc (138 mg, 98%). ¹H NMR (800 MHz,D₂O) δ 5.16 (d, J=3.2 Hz, 0.4H), 4.65 (d, J=8.0 Hz, 0.4H), 4.64 (d,J=8.0 Hz, 0.6H), 4.60 (d, J=8.0 Hz, 0.6H), 4.50 (d, J=8.0 Hz, 1H), 4.38(d, J=8.0 Hz, 1H), 4.10 (d, J=3.2 Hz, 1H), 4.06 (dd, J=3.2 and 9.6 Hz,1H), 3.91-3.21 (m, 29H), 2.70 (dd, J=4.8 and 12.8 Hz, 1H), 1.97 (s, 6H),1.74 (t, J=12.0 Hz, 1H). ¹³C NMR (200 MHz, D₂O) β-isomer: δ 174.86,174.77, 173.76, 102.78, 102.69, 102.38, 99.65, 95, 59, 81.90, 78.08,77.77, 75.29, 75.00, 74.73, 74.37, 74.17, 73.59, 72.70, 71.96, 71.60,71.22, 70.93, 69.81, 69.21, 68.20, 67.88, 67.28, 62.37, 60.87, 59.85,59.72, 59.61, 56.03, 51.58, 39.92, 22.02, 21.89. HRMS (ESI) m/zcalculated for C₃₇H₆₁N₂O₂₉ (M−H) 997.3360, found 997.3364.

Human Milk Oligosaccharides (HMOs) and Galactooligosaccharides (GOS).

Human milk was obtained from 36 healthy volunteers recruited at the UCSDMedical Center, San Diego, Calif., after approval by the University'sInstitute Review Board. Human milk oligosaccharides (HMOs) were isolatedfrom human milk as previously described. GOS syrup (Vivinal, dry matter75%) was kindly provided by Friesland Campina Domo, The Netherlands.

Rat Studies. The efficacy of the synthesized glycans against necrotizingenterocolitis was tested in the same neonatal rat model as previouslydescribed for DSLNT. Briefly, pregnant time-dated Sprague-Dawley ratswere induced at term and immediately randomized into one of thedifferent study groups. Animals in the dam-fed group (DF) remained withthe dam. All other animals were separated from the dam, housed in atemperature- and humidity-controlled incubator and orally gavaged with aspecial rodent formula (0.2 mL) twice daily. All animals, dam-fed andgavaged, were exposed to 10 min of hypoxia (5% O₂, 95% N₂) thrice dailyin a modular chamber. All animals were sacrificed 96 hours post-partum,and 0.5 cm of the terminal ileum prepared for H&E staining per standardprotocols and scored blindly based on morphological changes thatincluded epithelial sloughing, villus edema, infiltration ofneutrophils, apoptosis of villus enterocytes, crypt hyperplasia andmisaligned nuclei in the epithelium. If at least one pathology sign wasobserved, a score of 0.5-1.5 was assigned depending on severity. Two orthree signs together resulted in a score of 2-3. The maximum score of 4was given in case of complete obliteration of the epithelium with orwithout intestinal perforation. Pathology scores were plotted for eachanimal and the mean calculated per group. Differences between the groupswere calculated by Tway ANOVA with Kruskal-Wallis test and Dunn'sMultiple Comparison test. Significance was defined as a P value of lessthan 0.05.

Results

A general process for sequential one-pot multi-enzyme (OPME) reactionsis shown in FIG. 37. In nature, the key enzymes that catalyze theglycosidic bond formation are glycosyltransferases (GlyT).GlyT-catalyzed transfer of mammalian monosaccharides other than sialicacids can be achieved most efficiently via three steps in a salvagepathway: activation of a monosaccharide by a glycokinase (GlyK) to forma sugar-1-phosphate (monosaccharide-1-P), which can be used by anucleotidyltransferase (NucT) for the synthesis of a sugar nucleotide(or nucleotide diphosphate monosaccharide), the sugar nucleotide donorsubstrate of a suitable glycosyltransferase (GlyT) for the formation ofdesired glycosidic bond in the carbohydrate product. An inorganicpyrophosphorylase (PpA) can also be added to push the reaction towardscompletion in the direction of product formation. In comparison, moreenzymes are usually involved in longer processes for de novo pathwaysfor glycoside formation. Therefore, identifying suitableglycosyltransferases and the corresponding sugar nucleotide biosyntheticenzymes in the salvage pathways is important for developing efficientOPME systems. The availability, expression level, solubility, stabilityfor storage, and substrate promiscuity of enzymes are commonly evaluatedfor their application in large scale synthesis of carbohydrates andtheir structurally modified derivatives. Each OPME reaction can usuallybe used to add one monosaccharide to a glycosyltransferase acceptor.Carrying out the OPME reactions sequentially allows the formation ofcomplex carbohydrates and glycoconjugates. The stereo- andregio-specificities of the glycosidic bond formed, the nucleotidetriphosphate required, and the selection of related sugar nucleotidebiosynthetic enzymes are defined by the glycosyltransferases chosenbased on the structures of the desired carbohydrate products.

To obtain lacto-N-neotetraose (LNnT), a common human milktetrasaccharide (38-3), Lc₃ trisaccharide GlcNAcβ1-3Galβ1-4Glc (38-2)(FIG. 38) was synthesized from simple and inexpensive disaccharidelactose (38-1) and monosaccharide N-acetylglucosamine (GlcNAc) using aone-pot four-enzyme GlcNAc activation and transfer system containingBifidobacterium longum strain ATCC55813 N-acetylhexosamine-1-kinase(NahK), Pasteurella multocida N-acetylglucosamine uridyltransferase(PmGlmU), Pasteurella multocida inorganic pyrophosphatase (PmPpA), andNeisseria meningitidis acetylglucosaminyltransferase (NmLgtA). In thissystem, adenosine 5′-triphosphate (ATP) and GlcNAc were used byNahK-catalyzed reaction to form GlcNAc-1-P, which was used with uridine5′-triphosphate (UTP) by PmGlmU to form UDP-GlcNAc, the sugar nucleotidedonor for NmLgtA for the production of Lc₃ trisaccharide from lactose.All of the four enzymes were quite active in Tris-HCl buffer at pH 8.0and Lc₃ trisaccharide was obtained in an excellent yield (95%) byincubation at 37° C. for 2 days.

Taking advantage of a promiscuous Bifidobacterium longum UDP-sugarpyrophosphorylase (BLUSP) which can produce uridine 5′-diphosphategalactose (UDP-Gal) directly from UTP and galactose-1-phosphate(Gal-1-P), LNnT Galβ1-4GlcNAcβ1-3Galβ1-4Glc (38-3) was synthesized fromLc₃ trisaccharide (38-2) and a simple galactose (Gal) in an excellentyield (92%) using a one-pot four-enzyme Gal activation and transferreaction containing Escherichia coli galactokinase (EcGalK), BLUSP,PmPpA, and Neisseria meningitidis β1-4-galactosyltransferase (NmLgtB).This is an improved and a more direct and effective system compared toour previously reported OPME β1-4-galactosylation process which involvedthe formation of UDP-glucose (UDP-Glc) from glucose-1-phosphate(Glc-1-P) followed by C4-epimerization to produce UDP-Gal indirectly.

Sialylation of LNnT can be achieved by sialyltransferase(SiaT)-catalyzed reaction with in situ generation of sugar nucleotidedonor CMP-sialic acid catalyzed by a CMP-sialic acid synthetase (CSS).For the synthesis of sialosides containing the most common sialic acidform, N-acetylneuraminic acid (Neu5Ac), the one-pot two-enzymesialylation system is sufficient and the application of a sialic acidaldolase to generate Neu5Ac from its six-carbon precursorN-acetylmannosamine (ManNAc) is not necessary as the commercial pricesfor Neu5Ac and ManNAc are similar.

Initial sialylation of LNnT using Neu5Ac in a one-pot two-enzyme sialicacid activation and transfer system containing Neisseria meningitidisCMP-sialic acid synthetase (NmCSS) and Photobacterium damselaeα2-6-sialyltransferase (Pd2,6ST) with an Neu5Ac to LNnT ratio of 1.5 to1 produced an unexpected mixture of mono-sialylated and disialyl LNnT(DSLNnT) which were difficult to separate. Increasing the Neu5Ac to LNnTratio to 2.4 to 1 led to the complete formation of DSLNnT hexasaccharideNeu5Acα2-6Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc (38-4) with anexcellent yield (99%). Nuclear magnetic resonance (NMR) data confirmedthat Pd2,6ST does not only add a Neu5Ac α2-6-linked to the terminal Gal,it also adds an α2-6-linked Neu5Ac to the internal Gal residue in LNnT.As shown in Table 1, the attachment of Neu5Ac to the C-6 of the internalGal (Gal^(II)) and the terminal Gal (Gal^(IV)) in LNnT results insignificant downfield shifts of the substituted carbons (a downfieldshift of 2.39 ppm for the C-6 of Gal^(II) and a downfield shift of 2.52ppm for the C-6 of Gal^(IV) in DSLNnT. There are obvious interactions ofthe Neu5Ac residues and GlcNAc^(III) and Glc^(I) which result in asignificant downfield shift of 2.58 ppm for the C-4 of GlcNAc^(III) anda downfield shift of 1.55 ppm for the C-4 of Glc^(I). These unusualchemical shift changes seen in Neu5Acα2-6Gal sialosides are inaccordance with those observed for the glycans with same or similarstructural element. To our knowledge, this property of adding multipleNeu5Ac residues to both terminal and internal Gal by Pd2,6ST has neverbeen discovered before and the obtained DSLNnT is a new structure thathas never been synthesized.

TABLE 13 ¹³C NMR chemical shifts for compound Galβ1-4Glc (Lac),GlcNAcβ1-3Galβ1-4Glc (Lc₃ glycan), Galβ1-4GlcNAcβ1- 3Galβ1-4Glc (LNnT;38-3), and Neu5Acα2-6Galβ1-4GlcNAcβ1- 3(Neu5Acα2-6)Galβ1-4Glc (DSLNnT;38-4). VI IV III V II INeu5Acαa2-6Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc Carbon Lc₃ Sugar Unitatoms Lac glycan LNnT DSLNnT β-D-Glc^(I) 1 95.64 95.66 95.61 95.70 273.70 73.71 73.65 73.37 3 74.26 74.20 74.22 74.36 4 78.19 78.21 78.2179.76 5 74.69 74.71 74.76 74.76 6 59.78 60.01 60.34 60.23β-D-Gal^(II)(1-4) 1 102.79 102.84 102.76 103.35 2 70.86 70.03 69.8869.76 3 72.42 81.87 81.82 82.29 4 68.46 68.26 68.22 68.32 5 75.25 74.8075.52 73.77 6 60.94 60.88 60.84 63.23 β-D-GlcNAc^(III)(1-3) 1 102.75102.73 102.74 2 56.58 56.52 54.83 3 73.49 73.42 72.50 4 69.92 78.1180.69 5 75.57 74.66 74.72 6 60.41 59.93 60.05 C═O 174.87 174.83 175.01CH₃ 22.09 22.03 22.38 β-D-Gal^(IV)(1-4) 1 102.79 103.65 2 70.99 70.83 373.42 72.62 4 68.24 68.47 5 75.52 73.80 6 60.85 63.37α-D-Neu5Ac^(V)(2-6) 1 173.59 2 100.38 3 40.17 4 68.47 5 51.69 6 72.64 768.50 8 71.86 9 62.53 C═O 175.01 CH₃ 22.12 α-D-Neu5Ac^(VI)(2-6) 1 173.662 100.23 3 40.17 4 68.47 5 51.79 6 72.64 7 68.50 8 71.81 9 62.53 C═O175.01 CH₃ 22.15

As DSLNnT resembles some structural features of DSLNT, we hypothesizedthat the newly obtained DSLNnT may also have NEC preventing activity. Asa control, monosialyl pentasaccharide 3′″-sialyl LNnT (3′″-sLNnT) wassynthesized from LNnT using a one-pot two-enzyme sialic acid activationand transfer system similar to that described above for DSLNnT exceptthat a different sialyltransferase was used. A single-site mutant ofPasteurella multocida multifunctional α2-3-sialyltransferase (Pm STIM144D) was used to form an α2-3-sialyl linkage instead of theα2-6-sialyl linkages in DSLNnT described above. In addition, unlikePd2,6ST-catalyzed sialylation reaction which could add either one or twoα2-6-linked sialic acid residues to LNnT, PmST1 M144D-catalyzedsialylation reaction only added one α2-3-linked sialic acid residue tothe terminal Gal in LNnT. The application of the PmST1 M144D mutantinstead of wild-type PmST1 avoids the product hydrolysis by theα2-3-sialidase activity of the wild-type enzyme, thus improves the yieldof the one-pot two-enzyme α2-3-sialylation reaction. Indeed, anexcellent yield (98%) was achieved without the need of close monitor ofthe reaction process or to stop the reaction promptly.

The efficacy of DSLNnT or 3′″-sLNnT in protecting against NEC was testedin the same neonatal rat model that had been used previously to show theNEC protection effect of DSLNT. Since DSLNT was not available, a mixtureof human milk oligosaccharides (HMOs) isolated from pooled human milkwas used as a positive intervention control instead. Agalactooligosaccharides (GOS) sample, shown to be ineffective inpreventing NEC, was used as negative intervention control. As shown inFIG. 39, dam-fed (DF) animals hardly developed any signs of NEC (meanpathology score 0.67±0.34). Pathology scores were significantly higherin animals that were orally gavaged with rodent formula (FF) without theaddition of glycans (2.02±0.63, p<0.0001 compared to DF). Adding HMOs tothe formula led to significantly lower pathology scores (0.90±0.47,p<0.0001 compared to FF), which were not significantly different fromthe DF control (p=0.122). Adding GOS had no effect on lowering pathologyscores (2.00±0.64, p=0.915 compared to FF). All these results are inaccordance with the previously reported data. Adding the synthesizedDSLNnT to the formula led to significantly lower pathology scores(1.29±0.54, p=0.0008 compared to FF), which was not significantlydifferent from the effects seen in animals that received HMOs (p=0.052),but still different from the DF control (p=0.0013). Adding thesynthesized 3′″-sLNnT to the formula did not lower pathology scores(2.05±0.55, p=0.872 compared to FF).

These results show that similar to DSLNT, DSLNnT reduces pathologyscores in an NEC neonatal rat model. Both DSLNT and DSLNnT are disialylhexasaccharides but with noticeable structural differences. First ofall, DSLNT is a disialyl type I glycan whose core tetrasaccharide has aGal residue β1-3-linked to Lc₃ trisaccharide, while DSLNnT is a disialyltype II glycan whose core tetrasaccharide has a Gal residue β1-4-linkedto the Lc₃ trisaccharide. Secondly, although both have a Neu5Acα2-6-linked to an internal monosaccharide, the internal monosaccharideis GlcNAc in DSLNT while a Gal in DSLNnT. Thirdly, the outermost Neu5Acis linked to the penultimate Gal in an α2-3-linkage in DSLNT but anα2-6-linkage in DSLNnT. These structural differences of DSLNT and DSLNnTand their similarity in protecting neonatal rats from NEC indicate thatthe negatively charged disialyl component is important for the NECpreventing effect while the tetrasaccharide scaffold (type I or type II)does not seem to be important. The importance of disialyl component isfurther supported by the lacking of NEC preventing effect by monosialylpentasaccharides such as LSTb shown previously and 3′″-sLNnT shown here.

As DSLNnT is synthetically readily available, it has the potential to beused for treating NEC in preterm infants. The sequential OPME systemsdescribed here allow the use of inexpensive and simple disaccharide andmonosaccharides to synthesize desired complex oligosaccharides with highefficiency and selectivity. These are efficient approaches that can beused to obtain DSLNnT in amounts large enough for pre-clinical andclinical applications.

The novel synthetic disialyllacto-N-neotetraose (DSLNnT) can protectneonatal rats from NEC. Unlike the NEC-preventing DSLNT previouslyidentified from human milk which is not easily obtainable by eitherpurification or synthesis, the newly identified DSLNnT is readilyavailable by enzymatic synthesis. Highly efficient one-pot multienzymeGlcNAcylation, galactosylation, and sialylation systems for thehigh-yield synthesis of DSLNnT have been established. The readilyavailable DSLNnT is a good therapeutic candidate for treating NEC inpreterm infants.

Example 15 Synthesis of Other Novel Disialyl Oligosaccharides Compound15.2a: Neu5Acα2-3(Neu5Acα2-6)Galβ1-4Glc

Neu5Acα2-3Galβ1-4Glc (GM3, 83 mg, 0.13 mmol), Neu5Ac (61 mg, 0.20 mmol),and CTP (113 mg, 0.20 mmol) were dissolved in Tris-HCl buffer (5 mL, pH8.5) containing MgCl₂ (20 mM), N. meningitidis CMP-sialic acidsynthetase (NmCSS, 1.7 mg), and Photobacterium damselaeα2-6-sialyltransferase (Pd2,6ST, 2.1 mg). The reaction was carried outby incubating the reaction mixture in an incubator shaker at 37° C. for24 hrs. The reaction was monitored by TLC (n-PrOH:H₂O:NH₄OH=4:2:1 byvolume and detected by p-anisaldehyde sugar stain) and massspectrometry. When an optimal yield was achieved, to the reactionmixture was added the same volume (5 mL) of EtOH, and the mixture wasincubated at 4° C. for 30 min. The precipitates were removed bycentrifugation and the supernatant was concentrated and purified by aBio-Gel P-2 gel column (water was used as eluent). Further purificationwas achieved by silica gel chromatography (EtOAc:MeOH:H₂O=4:2:1 byvolume) and finally by Bio-Gel P-2 column (eluted with H₂O) to provideNeu5Acα2-3(Neu5Acα2-6)Galβ1-4Glc tetrasaccharide (112 mg, 93%). ¹H NMR(600 MHz, D₂O) δ 5.24 (d, J=3.6 Hz, 0.4H), 4.69 (d, J=7.8 Hz, 0.6H),4.53 (d, J=7.8 Hz, 1H), 4.15-4.13 (m, 1H), 4.00-3.96 (m, 3H), 3.91-3.62(m, 9H) 3.72-3.32 (m, 13H), 2.77 (dd, J=4.2 and 12.0 Hz, 1H), 2.72 (dd,J=4.2 and 12.0 Hz, 1H), 2.05 (s, 6H), 1.82 (t, J=12.0 Hz, 1H), 1.76 (t,J=12.0 Hz, 1H). ¹³C NMR (150 MHz, D₂O) β-isomer: δ 174.90, 174.79,173.73, 173.38, 102.87, 100.19, 99.90, 95.59, 79.67, 79.56, 75.18,74.57, 74.52, 73.63, 73.42, 72.78, 72.41, 71.65, 71.62, 69.16, 68.33,68.32, 68.02, 67.52, 63.45, 62.57, 62.50, 60.20, 51.69, 51.58, 39.98,39.40, 21.98, 21.96. HRMS (ESI) m/z calculated for C₃₄H₅₆N₂O₂₇ (M−H)924.3070, found 924.3060.

Compound 15.2b: Neu5Acα2-8Neu5Acα2-3 Galβ1-4Glc

Neu5Acα2-3Galβ1-4Glc (GM3, 200 mg, 0.32 mmol), Neu5Ac (107 mg, 0.35mmol), and CTP (197 mg, 0.35 mmol) were dissolved in Tris-HCl buffer (15mL, pH 8.5) containing MgCl₂ (20 mM), NmCSS (3.8 mg), and Campylobacterjejuni α2-8-sialyltransferase (CstII, 3.0 mg). The reaction was carriedout by incubating the reaction mixture in an incubator shaker at 37° C.for 20 hrs. The reaction was monitored by TLC (n-PrOH:H₂O:NH₄OH=4:2:1 byvolume and detected by p-anisaldehyde sugar stain) and massspectrometry. When an optimal yield was achieved, to the reactionmixture was added the same volume (15 mL) of EtOH, and the mixture wasincubated at 4° C. for 30 min. The precipitates were removed by thecentrifuge and the supernatant was concentrated and purified by aBio-Gel P-2 gel column (water was used as eluent). Further purificationwas achieved by silica gel chromatography (EtOAc:MeOH:H₂O=5:3:2 byvolume) and finally Bio-Gel P-2 column (eluted with H₂O) to provideNeu5Acα2-8Neu5Acα2-3Galβ1-4Glc tetrasaccharide (239 mg, 82%). ¹H NMR(800 MHz, D₂O) δ 5.22 (d, J=3.2 Hz, 0.3H), 4.66 (d, J=8.0 Hz, 0.7H),4.52 (d, J=7.2 Hz, 1H), 4.18-4.08 (m, 3H), 3.98-3.95 (m, 2H), 3.91-3.29(m, 21H), 2.77 (dd, J=4.8 and 12.8 Hz, 1H), 2.67 (dd, J=4.8 and 12.8 Hz,1H), 2.07 (s, 3H), 2.03 (s, 3H), 1.74 (t, J=12.0 Hz, 2H). ¹³C NMR (200MHz, D₂O) β-isomer: δ 174.89, 174.88, 173.40, 173.26, 102.58, 100.42,100.09, 95.71, 78.09, 77.98, 77.83, 75.34, 75.11, 74.72, 74.17, 73.90,72.54, 71.69, 71.08, 69.17, 68.37, 68.00, 67.81, 67.39, 62.49, 61.46,61.99, 59.89, 51.18, 51.66, 40.38, 39.61, 22.23, 21.96. HRMS (ESI) m/zcalculated for C₃₄H₅₆N₂O₂₇ (M−H) 924.3070, found 924.3071.

Compound 15.11: Neu5Acα2-6Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc(DSLNnT)

To prepare DSLNnT, a reaction mixture in a total volume of 10 mL inTris-HCl buffer (100 mM, pH 8.5) containing LNnT (131 mg, 0.19 mmol),Neu5Ac (143 mg, 0.46 mmol), CTP (260 mg, 0.46 mmol), MgCl₂ (20 mM),NmCSS (3.0 mg), and Pdα-2,6ST (2.0 mg) was incubated in a shaker at 37°C. for 36 hrs. The reaction was monitored by TLC (n-PrOH:H₂O:NH₄OH=4:2:1by volume and detected by p-anisaldehyde sugar stain) and massspectrometry. When an optimal yield was achieved, the reaction mixturewas added with the same volume (10 mL) of ethanol and incubated at 4° C.for 30 min. The precipitates were removed by centrifugation and thesupernatant was concentrated and purified by a Bio Gel P-2 gel column(water was used as an eluent). Further purification was achieved bysilica gel chromatography (EtOAc:MeOH:H₂O=4:3:2 by volume) and finallyBio-Gel P-2 column (eluted with H₂O) to produceNeu5Acα2-6Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc hexasaccharide (236mg, 99%). ¹H NMR (800 MHz, D₂O) δ 5.20 (d, J=4.0 Hz, 0.5H), 4.69 (d,J=8.0 Hz, 1H), 4.65 (d, J=8.0 Hz, 0.5H), 4.42 (d, J=8.0 Hz, 1H), 4.41(d, J=8.0 Hz, 1H), 4.17 (d, J=2.4 Hz, 1H), 3.99-3.28 (m, 37H), 2.68 (dd,J=4.8 and 12.8 Hz, 1H), 2.64 (dd, J=4.8 and 12.8 Hz, 1H), 2.03 (s, 3H),2.01 (s, 6H), 1.73-1.69 (m, 2H). ¹³C NMR (200 MHz, D₂O) β-isomer: δ175.01 (3C), 173.66, 173.59, 103.65, 103.35, 102.74, 100.38, 100.23,95.70, 82.29, 80.69, 79.76, 74.76, 74.72, 74.36, 73.80, 73.77, 73.37,72.64 (2C), 72.62, 72.50, 71.86, 71.81, 70.83, 69.76, 68.50 (2C), 68.47(3C), 68.32, 63.37, 63.23, 62.53 (2C), 60.23, 60.05, 54.83 (2C), 51.79,51.69, 40.17 (2C), 22.38, 21.15, 21.12. HRMS (ESI) m/z calculated forC₄₈H₇₈N₃O₃₇ (M−H) 1288.4314, found 1288.4305.

Compound 15.12: Neu5Acα2-6Galβ1-3GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc(DSLNT′)

Galβ1-3GlcNAcβ1-3Galβ1-4Glc (LNT, 150 mg, 0.21 mmol), Neu5Ac (169 mg,0.55 mmol), and CTP (307 mg, 0.55 mmol) were dissolved in Tris-HClbuffer (10 mL, pH 8.5) containing MgCl₂ (20 mM), NmCSS (4.0 mg), andPd2,6ST (3.0 mg). The reaction was carried out by incubating thereaction mixture in an incubator shaker at 37° C. for 36 hrs. Thereaction was monitored by TLC (n-PrOH:H₂O:NH₄OH=4:2:1 by volume anddetected by p-anisaldehyde sugar stain) and mass spectrometry. When anoptimal yield was achieved, to the reaction mixture was added the samevolume (10 mL) of EtOH and the mixture was incubated at 4° C. for 30min. The precipitates were removed by centrifugation and the supernatantwas concentrated and purified by a Bio-Gel P-2 gel column (water wasused as eluent). Further purification was achieved by silica gelchromatography (EtOAc:MeOH:H₂O=4:3:2 by volume) and finally Bio-Gel P-2column (eluted with H₂O) to provideNeu5Acα2-6Galβ1-3GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc hexasaccharide (268mg, 98%). ¹H NMR (600 MHz, D₂O) δ 5.24 (d, J=3.6 Hz, 0.4H), 4.74 (d,J=8.4 Hz, 1H), 4.68 (d, J=8.4 Hz, 0.6H), 4.44 (d, J=7.8 Hz, 1H), 4.40(d, J=7.8 Hz, 1H), 4.19 (d, J=3.8 Hz, 1H), 4.00-3.31 (m, 371-1),2.73-2.70 (m, 2H), 2.04 (s, 6H), 2.03 (s, 3H), 1.75 (t, J=12.0 Hz, 1H),1.71 (t, J=12.0 Hz, 1H). ¹³C NMR (200 MHz, D₂O) β-isomer: δ 174.86,174.83 (2C), 173.42 (2C), 103.80, 103.14, 102.51, 100.19, 100.07, 95.56,83.58, 81.98, 79.57, 75.19, 74.58, 74.54, 73.62, 73.49, 73.21, 72.43,72.38, 72.29, 71.74, 71.68, 70.45, 69.88, 69.66, 68.61, 68.38, 68.31,68.29, 68.20, 68.08, 63.41, 63.39, 62.56 (2C), 60.58, 60.17, 54.37,51.74, 51.70, 40.07, 40.02, 22.15, 21.98 (2C). HRMS (ESI) m/z calculatedfor C₄₈H₇₈N₃O₃₇ (M−H) 1288.4314, found 1288.4290.

Compound 15.18: Neu5Acα2-3(Neu5Acα2-6)Galβ1-9Kdn

Neu5Acα2-3Galβ1-9Kdn was prepared first by following the general one-pottwo-enzyme α2-3-sialylation system described above from Galβ1-9Kdn (101mg, 0.22 mmol) in Tris-HCl buffer (100 mM, pH 8.5) containing Neu5Ac(109 mg, 0.33 mmol), CTP (188 mg, 0.33 mmol), MgCl₂ (20 mM), NmCSS (0.8mg), and PmST1 (0.04 mg). The reaction was incubated at 37° C. for 3 hwith shaking (120 rpm). The crude product was purified by Bio-Gel P-2gel filtration to afford the sialoside product Neu5Acα2-3Galβ1-9Kdn (148mg, 89%). ¹H NMR (600 MHz, D₂O): δ 4.33 (d, 1H, J=7.8 Hz, H′-1), 4.00(dd, 1H, J=1.8 and 10.8 Hz), 3.93 (dd, 1H, J=3.6 and 11.2 Hz), 3.82-3.65(m, 9H), 3.60-3.39 (m, 9H), 2.58 (dd, 1H, J=4.2 and 12.6 Hz, H-3 eq″),2.01 (dd, 1H, J=4.8 and 13.2 Hz, H-3 eq), 1.85 (s, 3H, CH₃), 1.62 (t,1H, J=12.0 Hz, H-3ax), 1.60 (t, 1H, J=12.0 Hz, H-3ax″). ¹³C NMR (75 MHz,D₂O): δ 176.29, 175.17, 173.95, 103.28, 99.93, 96.35, 75.87, 75.07,73.02, 71.93, 71.90, 71.86, 71.61, 70.44, 69.64, 69.50, 69.15, 68.48,68.25, 67.97, 67.67, 62.75, 61.13, 59.50, 51.84, 39.82, 39.14, 22.22.HRMS (ESI) calcd. for C₂₆H₄₁NNaO₂₂ (MNa), 742.2018, found 742.2001.

Neu5Acα2-3(Neu5Acα2-6)Galβ1-9Kdn was then prepared fromNeu5Acα2-3Galβ1-9Kdn (130 mg, 0.17 mmol) in Tris-HCl buffer (100 mM, pH8.5) containing Neu5Ac (84 mg, 0.25 mmol), CTP (143 mg, 0.25 mmol),MgCl₂ (20 mM), NmCSS (0.8 mg), and Pd2,6ST (0.2 mg). The reaction wasincubated at 37° C. for 12 hrs with shaking (120 rpm). The crude productwas purified by Bio-Gel P-2 gel filtration to afford the sialosideproduct (159 mg, 87%). ¹H NMR (600 MHz, D₂O): δ 4.33 (d, 1H, J=7.8 Hz,H′-1), 3.99 (m, 1H), 3.94 (dd, 1H, J=3.0 and 9.6 Hz), 3.83-3.59 (m,13H), 3.56-3.38 (m, 12H), 2.59-2.53 (m, 2H, H-3 eq″, H-3 eq′″), 2.00(dd, 1H, J=4.8 and 13.2 Hz, H-3 eq), 1.85 (s, 6H, 2CH₃), 1.64-1.57 (m,2H, H-3ax, H-3ax″), 1.52 (t, 1H, J=12.0 Hz, H-3ax′″). ¹³C NMR (75 MHz,D₂O): δ 177.12, 175.16 (2C), 173.94, 173.60, 103.26, 100.64, 100.13,96.62, 75.66, 73.40, 73.02, 72.78, 71.96, 71.90, 71.87, 71.55, 70.59,69.49, 69.44, 69.27, 68.56, 68.49, 68.47, 68.28, 67.79, 63.57, 62.85,62.78, 61.03, 52.04, 51.84, 40.31, 39.73, 39.32, 22.27 (2C). HRMS (ESI)calcd. for C₃₇H₅₇N₂Na₂O₃₀ (M-Na) 1055.2792, found 1055.2853.

DNA AND PROTEIN SEQUENCES FOR GENES AND ENZYMESDNA sequence of NahK_ATCC15697 (SEQ ID NO: 1)(Note: The sequences for His₆-tag are underlined)ATGAACAACACCAATGAAGCCCTGTTCGACGTCGCTTCGCACTTCGCGCTGGAAGGCACCGTCGACAGCATCGAACCATACGGAGACGGCCATATCAACACCACCTATCTGGTGACCACGGACGGCCCCCGCTACATCCTCCAACGGATGAACACCGGCATCTTCCCCGATACGGTGAATCTGATGCGCAATGTCGAGCTGGTCACCTCCACTCTCAAGGCTCAGGGCAAAGAGACGCTGGACATCGTGCGCACCACCTCCGGCGACACCTGGGCCGAGATCGACGGCGGCGCATGGCGCGTCTACAAGTTCATCGAACACACCATGTCATACAACCTCGTGCCGAACCCGGACGTGTTCCGCGAAGCCGGCAGGGCGTTCGGTGATTTCCAGAACTTCCTGTCCGGGTTCGACGCCAACCAGCTGACCGAGACCATCGCCCACTTCCACGACACCCCGCACCGCTTCGAGGACTTCAAGAAGGCGCTCGCCGCGGACGAGCTCGGGCGTGCCGCCGGGTGCGGCCCGGAGATCGAGTTCTATCTGAGTCACGCCGACCAGTACGCCGTCGTGATGGATGGGCTCAGGGATGGTTCGATCCCGCTGCGCGTGACCCACAACGACACCAAACTCAACAACATCCTCATGGATGCCACCACCGGCAAGGCCCGTGCGATCATCGATCTAGACACCATCATGCCGGGGTCCATGCTCTTCGACTTCGGCGATTCCATCCGTTTCGGCGCGTCCACGGCCTTGGAGGATGAGCGGGATCTGGACAAGGTGCATTTCAGCACCGAGCTGTTCCGCGCCTACACGGAAGGCTTCGTGGGCGAACTACGCGACAGCATCACCGCGCGCGAGGCCGAACTGCTGCCGTTCAGCGGCAACCTGCTCACCATGGAATGCGGCATGCGCTTTCTCGCCGACTACCTGGAAGGCGACGTCTACTTCGCCACCAAGTACCCCGAGCATAACCTGGTGCGCTCCCGCACCCAGATCAAGCTCGTGAGGGAGATGGAGCAGCGAGCCGATGAGACCCGCGCCATCGTGGCCGACGTCATGGAGTCGACCAAGCTCGAGCACCACCACCACCACCACTGAProtein sequence of NahK_ATCC15697 (SEQ ID NO: 2)(Note: The sequences for His₆-tag are underlined)MNNTNEALFDVASHFALEGTVDSIEPYGDGHINTTYLVTTDGPRYILQRMNTGIFPDTVNLMRNVELVTSTLKAQGKETLDIVRTTSGDTWAEIDGGAWRVYKFIEHTMSYNLVPNPDVFREAGRAFGDFQNFLSGFDANQLTETIAHFHDTPHRFEDFKKALAADELGRAAGCGPEIEFYLSHADQYAVVMDGLRDGSIPLRVTHNDTKLNNILMDATTGKARAIIDLDTIMPGSMLFDFGDSIREGASTALEDERDLDKVHFSTELFRAYTEGFVGELRDSITAREAELLPFSGNLLTMECGMRFLADYLEGDVYFATKYPEHNLVRSRTQIKLVREMEQRADETRAIVADVMESTKLEHHHHHHDNA sequence of NahK_ATCC55813  (SEQ ID NO: 3)(Note: The sequences for His₆-tag are underlined)ATGACCGAAAGCAATGAAGTTTTATTCGGCATCGCCTCGCATTTTGCGCTGGAAGGTGCCGTGACCGGTATCGAACCTTACGGAGACGGCCACATCAACACCACCTATCTGGTGACCACGGACGGCCCCCGCTACATCCTCCAGCAGATGAACACCAGCATCTTCCCCGATACGGTGAATCTGATGCGCAATGTCGAACTGGTCACCTCCACTCTCAAGGCTCAGGGCAAAGAGACGCTGGACATTGTGCCCACCACCTCAGGCGCCACCTGGGCCGAGATCGATGGCGGCGCATGGCGCGTCTACAAGTTCATCGAACACACCGTGTCCTACAACCTCGTGCCGAACCCGGACGTGTTCCGCGAAGCCGGCAGCGCATTCGGCGACTTCCAGAACTTCCTGTCCGAATTCGACGCCAGCCAGCTGACCGAAACCATCGCCCACTTCCACGACACCCCGCATCGTTTCGAGGACTTCAAGGCCGCCCTCGCCGCGGACAAGCTCGGCCGCGCCGCCGCATGCCAGCCGGAAATCGACTTCTATCTGAGTCACGCCGACCAGTATGCCGTCGTGATGGATGGGCTCAGGGACGGTTCGATTCCGCTGCGCGTGACCCACAATGACACCAAGCTCAACAACATCCTCATGGACGCCACCACCGGCAAGGCGCGTGCGATCATCGATCTCGACACCATCATGCCCGGCTCCATGCTGTTCGACTTCGGCGATTCCATACGCTTTGGTGCGTCCACTGCTCTGGAAGACGAAAAGGACCTCAGCAAGGTGCATTTCAGCACCGAGCTGTTCCGCGCCTACACGGAAGGCTTCGTGGGCGAACTACGCGGCAGCATCACCGCGCGCGAGGCCGAACTGCTGCCGTTCAGCGGCAACCTGCTCACCATGGAATGCGGCATGCGCTTTCTCGCCGACTACTTGGAAGGCGATATCTACTTTGCCACCAAGTACCCCGAGCATAATCTGGTGCGCACCCGCACCCAGATCAAACTCGTGCAGGAGATGGAGCAGAAGGCCAGTGAAACCCACGCCATCGTAGCCGACATCATGGAGGCTGCCAGGCTCGAGCACCACCACCACCACCACTGAProtein sequence of NahK_ATCC55813  (SEQ ID NO: 4)(Note: The sequences for His₆-tag are underlined)MTESNEVLFGIASHFALEGAVTGIEPYGDGHINTTYLVTTDGPRYILQQMNTSIFPDTVNLMRNVELVTSTLKAQGKETLDIVPTTSGATWAEIDGGAWRVYKFIEHTVSYNLVPNPDVFREAGSAFGDFQNFLSEFDASQLTETIAHFHDTPHRFEDFKAALAADKLGRAAACQPEIDFYLSHADQYAVVMDGLRDGSIPLRVTHNDTKLNNILMDATTGKARAIIDLDTIMPGSMLFDFGDSIRFGASTALEDEKDLSKVHFSTELFRAYTEGFVGELRGSITAREAELLPFSGNLLTMECGMRFLADYLEGDIYFATKYPEHNLVRTRTQIKLVQEMEQKASETHAIVADIMEAARLEHHHHHHDNA sequence of AtGlcAK (SEQ ID NO: 5)(Note: Italic sections of the sequences are from pET15b vectorand primer).ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGATCCGAATTCCACGGTTTCCGGCGATGGTCAGGCGACGGCGGCGATAGAGCATCGGTCCTTCGCTCGGATCGGATTTCTCGGAAACCCGAGCGATGTATACTTCGGGCGAACCATATCATTGACCATCGGAAACTTCTGGGCATCCGTGAAGCTGGAGCCATCGGAGCATCTCGTAATCAAGCCTCATCCATTCCATGATCTCGTCCAGTTCACCTCTCTCGACCATCTCCTGAATCGTTTGCAAAATGAAGGATACTACGGTGGGGTAAGGTTGCTAATGGCGATATGTAAAGTATTCCGTAACTATTGCAAAGAGAATGACATTCAACTTCACCAAGCCAACTTCTCTCTTTCTTATGATACCAATATCCCTAGGCAGACAGGGCTTTCGGGTTCTAGTGCCATCGTATCCGCTGCCCTTAACTGCCTTCTCGACTTCTACAATGTCAGGCATTTGATCAAAGTACAAGTCCGCCCTAACATTGTTCTCAGTGCTGAGAAAGAACTTGGCATTGTTGCTGGTCTTCAGGACAGGGTTGCTCAGGTCTATGGTGGTCTTGTTCACATGGATTTTAGCAAGGAGCACATGGATAAATTGGGGCATGGGATTTACACTCCTATGGATATCAGTCTCCTCCCTCCTCTGCATCTCATCTATGCTGAGAATCCGAGCGACTCAGGGAAGGTACATAGTATGGTTCGGCAAAGATGGTTAGACGGTGATGAGTTTATAATCTCATCAATGAAAGAAGTCGGAAGTCTAGCAGAAGAAGGTCGAACTGCATTACTCAACAAGGACCATTCCAAACTTGTGGAACTCATGAACCTTAATTTCGACATTCGGAGGCGGATGTTTGGGGATGAATGCTTAGGAGCAATGAACATGGAGATGGTGGAAGTAGCAAGGAGGGTTGGTGCAGCCTCAAAGTTCACTGGAAGTGGAGGAGCAGTGGTGGTTTTCTGCCCTGAAGGTCCATCTCAGGTGAAACTTCTGGAAGAAGAATGCAGGAAAGCGGGATTTACGCTTCAGCCGGTAAAAATTGCGCCTTCATGTTTGAATGATTCTGACATTCAGACCTTATGAProtein sequence of AtGlcAK (SEQ ID NO: 6)(Note: Italic sectionsof the sequences are from pET15b vector and primer. N-terminalHis₆-tag (SEQ ID NO: 22) is underlined in the protein sequence) MGSSHHHHHH SSGLVPRGSHMDPNSTVSGDGQATAAIEHRSFARIGFLGNPSDVYFGRTISLTIGNFWASVKLEPSEHLVIKPHPFHDLVQFTSLDHLLNRLQNEGYYGGVRLLMAICKVFRNYCKENDIQLHQANFSLSYDTNIPRQTGLSGSSAIVSAALNCLLDFYNVRHLIKVQVRPNIVLSAEKELGIVAGLQDRVAQVYGGLVHMDFSKEHMDKLGHGIYTPMDISLLPPLHLIYAENPSDSGKVHSMVRQRWLDGDEFIISSMKEVGSLAEEGRTALLNKDHSKLVELMNLNFDIRRRMFGDECLGAMNMEMVEVARRVGAASKFTGSGGAVVVFCPEGPSQVKLLEEECRKAGFTLQPVKIAPSCLNDSDIQTL DNA sequence of His₆-PmGlmU (SEQ ID NO: 7)(Note: Italic sections of the sequences are from pET15bvector and primer)ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGAAAGAGAAAGCATTAAGTATCGTGATTTTAGCGGC A GGTAAAGGGACGCGGATGTATTCTGATTTACCAAAAGTGCTACATAAAATTGCCGGAAAACCGATGGTAAAACATGTGATCGATACGGTGAAATCCATTCATGCAAAAAATATCCATTTAGTGTATGGACATGGTGGGGAAGTGATGCA AACTCG CTTGCAAGATGAACCTGTGAATTGGGTCTTACAAGCCGAGCAATTAGGTACGGGGCATGCTATGCAGCAAGCAGCCCCGTTTTTTGCAGATGATGAAAATATTTTGATGCTTTATGGTGATGGACCATTAATTACTGC GA AAAC C TTACAAACATTAATTGCGGCAAAACCTGAACATGGTATTGCATTATTGACCGTCGTATTAGATGACCCAACTGGTTATGGGCGTATTGTGCGTGAAAATGGCAATGTGGTGGCGATTGTGGAACAAAAAGATGCCAATGCAGAGCAATTAAAAATCCAAGAAATTAACACAGGCTTGTTAGTGGCAGACGGTAAAAGTTTGAAAAAATGGTTATCACAGTTAACCAACAACAATGCACAGGGAGAATATTATATTACGGATGTGATCGCCTTAGCGAATCAAGACGGTTGCCA A GTAGTGGCGGTACAAGCCAGT A ACTTTATGGAAGTAGAGGGCGTGAATAACCGTCAGCAATTAGCGCGTTTAGAGCGTTATTATCAGCGCAAACAAGCAGACAATTTATTATTGGCTGGGGTGGCATTAGCGGATCCTGAGCGTTTTGATTTACGCGGGGAACTAAGCCATGGGA AAGACGTGC AAATTGATGTGAACGTGATTATCGAGGGCAAAGTCAGCTTAGGTCACCGAGTT AAAATTGGAGC AGGTTGTGTGTTAAAAAATTGCCAGATTGGTGATGATGTAGAAATTAAACCTTATTCTGTGTTGGAAGAGGCGATTGTTGGACAAGCTGCGCAAATTGGACCCTTCTCTCGTTTGCGTCCGGGG G C T GCATTAGCCGACAACACTCATATTGGTAATTTCGTTGAAATTAAGAAAGCGCATATTGGGAC A GGCTCGAAAGTAAACCATTTAAGTTATGTGGGAGATGCCGAAGTCGGGATGCAATGTAATATTGGTGCCGGCGTGATCACTTGTAACTATGATGGCGCAAATAAATTTAAGACCATTATTGGTGATAATGTGTTTGTAGGGTCTGATGTACAACTCGTGGCACCGGTTACCATCGAAACGGGTGCAACCATTGG T GCGGGGACTACGGTGACCAAAGATGTGGCTTGTGATGAGTTAGTGATTTCACGTGTTCCTCAACGTCATATTCAAGGTTGGCAACGCCCTACTAAACAAACGAAAAAGTAA Protein sequence of His₆-PmGlmU (SEQ ID NO: 8) (Note: Italicsections of the sequences are from pET15b vector and primer. N-terminal His₆-tag (SEQ ID NO: 22)is underlined in the proteinsequence) MGSS HHHHHHSSGLVPRGSHMKEKALSIVILAAGKGTRMYSDLPKVLHKIAGKPMVKHVIDTVKSIHAKNIHLVYGHGGEVMQTRLQDEPVNWVLQAEQLGTGHAMQQAAPFFADDENILMLYG DGPLITA KTLQTLIAAKPEHGIALLTVVLDDPTGYGRIVRENGNVVAIVEQKDANAEQLKIQEINTGLLVADGKSLKKWLSQLTNNNAQGEYYITDVIALANQDGCQVVAVQAS N FMEVEGVNNRQQLARLERYYQRKQADNLLLAGVALADPERFDLRGELSHGKDV Q IDVNVIIEGKVSLGHRVKIGAGCVLKNCQIGDDVEIKPYSVLEEAIVGQAAQIGPFSRLRPG A ALADNTHIGNFVEIKKAHIGTGSKVNHLSYVGDAEVGMQCNIGAGVITCNYDGANKFKTIIGDNVFVGSDVQLVAPVTIETGATIGAGTTVTKDVACDELVISRVPQRHIQGWQRPTKQTKKDNA sequence of BLUSP (SEQ ID NO: 9)(Note: The sequences for His₆-tag are underlined)ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGACAGAAATAAACGATAAGGCCCAACTGGATATCGCCGCCGCCGGCGACACCGACGCCGTTACCTCGGACACCCCCGAAGAAACCGTAAACACCCCCGAAGTGGATGAGACTTTCGAGCTTTCGGCCGCCAAGATGCGCGAGCATGGCATGAGCGAAACCGCCATCAACCAGTTCCACCATTTGTATGACGTATGGCGCCATGAAGAAGCCTCCAGCTGGATTCGTGAGGACGACATCGAGCCGCTTGGCCACGTGCCCAGCTTCCACGACGTCTATGAGACCATCAACCACGACAAGGCCGTGGACGCCTTCGCCAAGACCGCATTCCTCAAGCTCAATGGCGGTCTGGGCACCTCCATGGGATTAGACAAGGCCAAGTCGCTGTTGCCGGTGCGTAGGCACAAGGCCAAGCAGATGCGCTTCATCGACATCATCATCGGTCAGGTGCTTACCGCTCGCACCCGCCTGAACGTCGAACTGCCGCTGACGTTCATGAACTCCTTCCACACTTCGGCGGACACGATGAAGGTGCTCAAGCATCATCGCAAGTTCAGTCAGCATGACGTGCCGATGGAAATCATCCAGCATCAGGAACCCAAGCTCGTGGCCGCCACCGGCGAACCTGTGAGCTACCCTGCGAACCCGGAGCTGGAATGGTGCCCGCCCGGCCACGGCGACCTGTTCTCCACCATCTGGGAGTCTGGTCTGCTTGACGTATTGGAGGAGCGCGGCTTCAAGTATCTGTTCATCTCCAATTCCGACAATCTCGGTGCGCGCGCCTCGCGTACGTTGGCCCAGCACTTCGAAAACACAGGTGCCCCGTTTATGGCTGAAGTGGCCATCCGCACCAAGGCCGATCGCAAGGGCGGCCATATTGTACGAGACAAGGCCACTGGTCGCCTAATACTGCGTGAAATGAGCCAGGTCCATCCGGACGATAAGGAAGCGGCCCAAGACATCACCAAGCATCCTTACTTCAACACCAACTCAATCTGGGTTCGCATCGACGCTTTGAAAGACAAGCTCGCCGAATGCGATGGTGTGTTGCCGTTGCCGGTGATTCGTAACAAAAAGACCGTGAATCCCACGGACCCGGATTCCGAACAGGTGATTCAGCTGGAAACCGCCATGGGCGCCGCAATCGGTCTGTTCAACGGTTCTATCTGCGTCCAAGTGGATCGTATGCGCTTCCTTCCGGTGAAAACCACCAATGATTTGTTCATTATGCGTTCCGATCGATTCCACCTGACGGACACGTATGAGATGGAAGACGGCAATTACATCTTCCCGAACGTCGAACTTGATCCGCGATACTACAAGAACATCCACGATTTCGACGAACGGTTCCCCTACGCCGTGCCATCTTTGGCCGCAGCCAACTCGGTTTCCATTCAGGGCGACTGGACATTCGGACGTGACGTCATGATGTTCGCCGACGCCAAACTGGAAGATAAAGGCGAGCCAAGCTATGTGCCGAACGGCGAATACGTTGGTCCGCAAGGCATCGAACCGGACGATTGGGTGTGAProtein sequence of BLUSP (SEQ ID NO: 10) (Note: The sequencesfor His₆-tag are underlined)MGSSHHHHHHSSGLVPRGSHMTEINDKAQLDIAAAGDTDAVTSDTPEETVNTPEVDETFELSAAKMREHGMSETAINQFHHLYDVWRHEEASSWIREDDIEPLGHVPSFHDVYETINHDKAVDAFAKTAFLKLNGGLGTSMGLDKAKSLLPVRRHKAKQMRFIDIIIGQVLTARTRLNVELPLTFMNSFHTSADTMKVLKHHRKFSQHDVPMEIIQHQEPKLVAATGEPVSYPANPELEWCPPGHGDLFSTIWESGLLDVLEERGFKYLFISNSDNLGARASRTLAQHFENTGAPFMAEVAIRTKADRKGGHIVRDKATGRLILREMSQVHPDDKEAAQDITKHPYFNTNSIWVRIDALKDKLAECDGVLPLPVIRNKKTVNPTDPDSEQVIQLETAMGAAIGLFNGSICVQVDRMRFLPVKTTNDLFIMRSDRFHLTDTYEMEDGNYIFPNVELDPRYYKNIHDFDERFPYAVPSLAAANSVSIQGDWTFGRDVMMFADAKLEDKGEPSYVPNGEYVGPQGIEPDDWVDNA sequence of PmUgd-His₆ (SEQ ID NO: 11) (Note: The sequencesfor His6-tag are underlined)ATGAAGAAAATTACAATTGCTGGGGCTGGCTATGTTGGTTTATCCAATGCAGTATTATTAGCTCAACACCACAATGTGATCTTATTAGATATTGATCAAAATAAAGTTGATTTAATTAATAATAAAAAATCGCCCATCACAGATAAAGAAATCGAAGATTTCTTACAAAATAAATCACTGACAATGATGGCAACAACAGATAAAGAAGTGGCATTAAAAAACGCAGACTTTGTCATCATCGCAACGCCAACAGACTATAATACCGAAACAGGTTATTTTAATACATCCACTGTTGAAGCTGTCATTGAACAAACCCTTTCAATCAATCCACAAGCAACGATTATTATAAAATCAACGATTCCCGTTGGTTTTACCGAAAAAATGCGTGAGAAATTTCATACCAAGAACATTATTTTTTCTCCTGAGTTTTTAAGAGAAGGAAAAGCACTTCATGACAATTTGTTTCCAAGCAGAATTATTGTTGGCAGTACTTCTTATCAAGCAAAAGTATTTGCCGATATGTTGACACAGTGTGCCAGAAAAAAAGATGTAACTGTTTTATTTACACACAATACTGAGGCTGAAGCTGTTAAATTATTTGCAAATACGTATCTCGCAATGCGAGTTGCCTTTTTTAATGAATTAGATACTTATGCGAGTCTTCACCATTTAAATACAAAAGACATTATCAATGGTATTTCTACTGATCCTCGCATTGGTACACACTACAATAACCCAAGTTTCGGCTATGGCGGTTATTGTTTACCCAAAGACACTAAACAGTTACTGGCTAACTATGCTGACGTACCTCAAAATCTCATTGAAGCCATTGTCAAATCTAATGAAACCAGAAAACGTTTCATTACTCATGATGTATTAAATAAGAAACCTAAAACTGTTGGTATTTATCGTTTAATCATGAAGTCAGGTTCTGATAACTTCAGAGCTTCTGCTATTCTCGATATTATGCCGCATCTCAAAGAAAACGGTGTTGAGATTGTGATTTATGAGCCAACCTTAAATCAACAGGCATTTGAGGACTACCCCGTTATTAATCAACTCTCTGAATTTATTAATCGCTCTGATGTCATTCTCGCTAATCGTTCTGAGCCAGATTTAAATCAATGTTCCCATAAAATCTATACAAGAGATATTTTTGGCGGTGATGCTCTCGAGCACCACCACCACCACCACTGAProtein sequence of PmUgd-His₆ (SEQ ID NO: 12) (Note: Thesequences for His₆-tag are underlined)MKKITIAGAGYVGLSNAVLLAQHHNVILLDIDQNKVDLINNKKSPITDKEIEDFLQNKSLTMMATTDKEVALKNADFVIIATPTDYNTETGYFNTSTVEAVIEQTLSINPQATIIIKSTIPVGFTEKMREKFHTKNIIFSPEFLREGKALHDNLFPSRIIVGSTSYQAKVFADMLTQCARKKDVTVLFTHNTEAEAVKLFANTYLAMRVAFFNELDTYASLHHLNTKDIINGISTDPRIGTHYNNPSFGYGGYCLPKDTKQLLANYADVPQNLIEAIVKSNETRKRFITHDVLNKKPKTVGIYRLIMKSGSDNFRASAILDIMPHLKENGVEIVIYEPTLNQQAFEDYPVINQLSEFINRSDVILANRSEPDLNQCSHKIYTRDIFGGDALEHHHHHHDNA sequence of MBP-PmHS1-His₆ (SEQ ID NO: 13) (Note: Italicsections of the sequences are from pMAL-c4X vector and primer.The sequences for His₆-tag are underlined)CTCGGGATCGAGGGAAGGATTTCAGAATTCGGATCCATGAGCTTATTTAAACGTGCTACTGAGCTATTTAAGTCAGGAAACTATAAAGATGCACTAACTCTATATGAAAATATAGCTAAAATTTATGGTTCAGAAAGCCTTGTTAAATATAATATTGATATATGTAAAAAAAATATAACACAATCAAAAAGTAATAAAATAGAAGAAGATAATATTTCTGGAGAAAACAAATTTTCAGTATCAATAAAAGATCTATATAACGAAATAAGCAATAGTGAATTAGGGATTACAAAAGAAAGACTAGGAGCCCCCCCTCTAGTCAGTATTATAATGACTTCTCATAATACAGAAAAATTCATTGAAGCCTCAATTAATTCACTATTATTGCAAACATACAATAACTTAGAAGTTATCGTTGTAGATGATTATAGCACAGATAAAACATTTCAGATCGCATCCAGAATAGCAAACTCTACAAGTAAAGTAAAAACATTCCGATTAAACTCAAATCTAGGGACATACTTTGCGAAAAATACAGGAATTTTAAAGTCTAAAGGAGATATTATTTTCTTTCAGGATAGCGATGATGTATGTCACCATGAAAGAATCGAAAGATGTGTTAATGCATTATTATCGAATAAAGATAATATAGCTGTTAGATGTGCATATTCTAGAATAAATCTAGAAACACAAAATATAATAAAAGTTAATGATAATAAATACAAATTAGGATTAATAACTTTAGGCGTTTATAGAAAAGTATTTAATGAAATTGGTTTTTTTAACTGCACAACCAAAGCATCGGATGATGAATTTTATCATAGAATAATTAAATACTATGGTAAAAATAGGATAAATAACTTATTTCTACCACTGTATTATAACACAATGCGTGAAGATTCATTATTTTCTGATATGGTTGAGTGGGTAGATGAAAATAATATAAAGCAAAAAACCTCTGATGCTAGACAAAATTATCTCCATGAATTCCAAAAAATACACAATGAAAGGAAATTAAATGAATTAAAAGAGATTTTTAGCTTTCCTAGAATTCATGACGCCTTACCTATATCAAAAGAAATGAGTAAGCTCAGCAACCCTAAAATTCCTGTTTATATAAATATATGCTCAATACCTTCAAGAATAAAACAACTTCAATACACTATTGGAGTACTAAAAAACCAATGCGATCATTTTCATATTTATCTTGATGGATATCCAGAAGTACCTGATTTTATAAAAAAACTAGGGAATAAAGCGACCGTTATTAATTGTCAAAACAAAAATGAGTCTATTAGAGATAATGGAAAGTTTATTCTATTAGAAAAACTTATAAAGGAAAATAAAGATGGATATTATATAACTTGTGATGATGATATCCGGTATCCTGCTGACTACATAAACACTATGATAAAAAAAATTAATAAATACAATGATAAAGCAGCAATTGGATTACATGGTGTTATATTCCCAAGTAGAGTCAACAAGTATTTTTCATCAGACAGAATTGTCTATAATTTTCAAAAACCTTTAGAAAATGATACTGCTGTAAATATATTAGGAACTGGAACTGTTGCCTTTAGAGTATCTATTTTTAATAAATTTTCTCTATCTGATTTTGAGCATCCTGGCATGGTAGATATCTATTTTTCTATACTATGTAAGAAAAACAATATACTCCAAGTTTGTATATCACGACCATCGAATTGGCTAACAGAAGATAACAAAAACACTGAGACCTTATTTCATGAATTCCAAAATAGAGATGAAATACAAAGTAAACTCATTATTTCAAACAACCCTTGGGGATACTCAAGTATATATCCATTATTAAATAATAATGCTAATTATTCTGAACTTATTCCGTGTTTATCTTTTTATAACGAGCATCATCATCATCATCACTAAProtein sequence of MBP-PmHS1-His₆ (SEQ ID NO: 14) (Note: Italicsections of the sequences are from pMAL-c4X vector and primer.The sequences for His₆-tag (SEQ ID NO: 22) are underlined)LGIEGRISEFGSMSLFKRATELFKSGNYKDALTLYENIAKIYGSESLVKYNIDICKKNITQSKSNKIEEDNISGENKFSVSIKDLYNEISNSELGITKERLGAPPLVSIIMTSHNTEKFIEASINSLLLQTYNNLEVIVVDDYSTDKTFQIASRIANSTSKVKTFRLNSNLGTYFAKNTGILKSKGDIIFFQDSDDVCHHERIERCVNALLSNKDNIAVRCAYSRINLETQNIIKVNDNKYKLGLITLGVYRKVFNEIGFFNCTTKASDDEFYHRIIKYYGKNRINNLFLPLYYNTMREDSLFSDMVEWVDENNIKQKTSDARQNYLHEFQKIHNERKLNELKEIFSFPRIHDALPISKEMSKLSNPKIPVYINICSIPSRIKQLQYTIGVLKNQCDHFHIYLDGYPEVPDFIKKLGNKATVINCQNKNESIRDNGKFILLEKLIKENKDGYYITCDDDIRYPADYINTMIKKINKYNDKAAIGLHGVIFPSRVNKYESSDRIVYNFQKPLENDTAVNILGTGTVAFRVSIFNKFSLSDFEHPGMVDIYFSILCKKNNILQVCISRPSNWLTEDNKNTETLFHEFQNRDEIQSKLIISNNPWGYSSIYPLLNNNANYSELIPCLSFYNEHHHHHHDNA sequence of His₆-PmHS2 (SEQ ID NO: 15) (Note: Italic sectionsof the sequences are from pET15b vector and primer. The sequences for His₆-tag are underlined) ATGGGCAGCAGC CATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGAAGGGAAAAAAAGAGATGACTCAAAAACAAATGACTAAAAATCCACCCCAACATGAAAAAGAAAATGAACTCAACACCTTTCAAAATAAAATTGATAGTCTAAAAACAACTTTAAACAAAGACATTATTTCTCAACAAACTTTATTGGCAAAACAGGACAGTAAACATCCGCTATCCGAATCCCTTGAAAACGAAAATAAACTTTTATTAAAACAACTCCAATTGGTTCTACAAGAATTTGAAAAAATATATACCTATAATCAAGCATTAGAAGCAAAGCTAGAAAAAGATAAGCAAACAACATCAATAACAGATTTATATAATGAAGTCGCTAAAAGTGATTTAGGGTTAGTCAAAGAAACCAACAGCGCAAATCCATTAGTCAGTATTATCATGACATCTCACAATACAGCGCAATTTATCGAAGCTTCTATTAATTCATTATTGTTACAAACATATAAAAACATAGAAATTATTATTGTAGATGATGATAGCTCGGATAATACATTTGAAATTGCCTCGAGAATAGCGAATACAACAAGCAAAGTCAGAGTATTTAGATTAAATTCAAACCTAGGAACTTACTTTGCGAAAAATACAGGCATATTAAAATCTAAAGGTGACATTATTTTCTTTCAAGATAGTGATGATGTATGTCATCATGAAAGAATAGAAAGATGTGTAAATATATTATTAGCTAATAAAGAAACTATTGCTGTTCGTTGTGCATACTCAAGACTAGCACCAGAAACACAACATATCATTAAAGTCAATAATATGGATTATAGATTAGGTTTTATAACCTTGGGTATGCACAGAAAAGTATTTCAAGAAATTGGTTTCTTCAATTGTACGACTAAAGGCTCAGATGATGAGTTTTTTCATAGAATTGCGAAATATTATGGAAAAGAAAAAATAAAAAATTTACTCTTGCCGTTATACTACAACACAATGAGAGAAAACTCTTTATTTACTGATATGGTTGAATGGATAGACAATCATAACATAATACAGAAAATGTCTGATACCAGACAACATTATGCAACCCTGTTTCAAGCGATGCATAACGAAACAGCCTCACATGATTTCAAAAATCTTTTTCAATTCCCTCGTATTTACGATGCCTTACCAGTACCACAAGAAATGAGTAAGTTGTCCAATCCTAAGATTCCTGTTTATATCAATATTTGTTCTATTCCCTCAAGAATAGCGCAATTACAACGTATTATCGGCATACTAAAAAATCAATGTGATCATTTTCATATTTATCTTGATGGCTATGTAGAAATCCCTGACTTCATAAAAAATTTAGGTAATAAAGCAACCGTTGTTCATTGCAAAGATAAAGATAACTCCATTAGAGATAATGGCAAATTCATTTTACTGGAAGAGTTGATTGAAAAAAATCAAGATGGATATTATATAACCTGTGATGATGACATTATCTATCCAAGCGATTACATCAATACGATGATCAAAAAGCTGAATGAATACGATGATAAAGCGGTTATTGGTTTACACGGCATTCTCTTTCCAAGTAGAATGACCAAATATTTTTCGGCGGATAGACTGGTATATAGCTTCTATAAACCTCTGGAAAAAGACAAAGCGGTCAATGTATTAGGTACAGGAACTGTTAGCTTTAGAGTCAGTCTCTTTAATCAATTTTCTCTTTCTGACTTTACCCATTCAGGCATGGCTGATATCTATTTCTCTCTCTTGTGTAAGAAAAATAATATTCTTCAGATTTGTATTTCAAGACCAGCAAACTGGCTAACGGAAGATAATAGAGACAGCGAAACACTCTATCATCAATATCGAGACAATGATGAGCAACAAACTCAGCTGATCATGGAAAACGGTCCATGGGGATATTCAAGTATTTATCCATTAGTCAAAAATCATCCTAAATTTACTGACCTTATCCCCTGTTTACCTTTTTATTTTTTATAAProtein sequence of His₆-PmHS2 (SEQ ID NO: 16) (Note: Italicsections of the sequences are from pET15b vector and primer.The sequences for His₆-tag (SEQ ID NO: 22) are underlined) MGSS HHHHHHSSGLVPRGSHMKGKKEMTQKQMTKNPPQHEKENELNTFQNKIDSLKTTLNKDIISQQTLLAKQDSKHPLSESLENENKLLLKQLQLVLQEFEKIYTYNQALEAKLEKDKQTTSITDLYNEVAKSDLGLVKETNSANPLVSIIMTSHNTAQFIEASINSLLLQTYKNIEIIIVDDDSSDNTFEIASRIANTTSKVRVFRLNSNLGTYFAKNTGILKSKGDIIFFQDSDDVCHHERIERCVNILLANKETIAVRCAYSRLAPETQHIIKVNNMDYRLGFITLGMHRKVFQEIGFFNCTTKGSDDEFFHRIAKYYGKEKIKNLLLPLYYNTMRENSLFTDMVEWIDNHNIIQKMSDTRQHYATLFQAMHNETASHDFKNLFQFPRIYDALPVPQEMSKLSNPKIPVYINICSIPSRIAQLQRIIGILKNQCDHFHIYLDGYVEIPDFIKNLGNKATVVHCKDKDNSIRDNGKFILLEELIEKNQDGYYITCDDDIIYPSDYINTMIKKLNEYDDKAVIGLHGILFPSRMTKYFSADRLVYSFYKPLEKDKAVNVLGTGTVSFRVSLENQFSLSDFTHSGMADIYESLLCKKNNILQICISRPANWLTEDNRDSETLYHQYRDNDEQQTQLIMENGPWGYSSIYPLVKNHPKFTDLIPCLPFYFLDNA sequence of MBP-KfiA-His₆ (SEQ ID NO: 17) (Note: Italicsections of the sequences are from pMAL-c4X vector and primer.The sequences for  His₆-tag are underlined)AACCTCGGGATCGAGGGAAGGATTTCAGAATTCATGATTGTTGCAAATATGAGCAGCTATCCTCCGCGTAAAAAAGAACTGGTTCATAGCATTCAGAGCCTGCATGCACAGGTGGATAAAATTAATCTGTGCCTGAATGAATTTGAAGAAATTCCGGAAGAACTGGATGGCTTTAGCAAACTGAATCCGGTTATTCCGGATAAAGATTATAAAGATGTGGGCAAATTTATTTTTCCGTGCGCCAAAAATGATATGATTGTTCTGACCGATGATGATATTATTTATCCGCCAGATTATGTGGAAAAAATGCTGAATTTTTATAATAGCTTTGCCATTTTTAATTGCATTGTGGGTATTCATGGCTGCATTTATATTGATGCCTTTGATGGTGATCAGAGCAAACGTAAAGTGTTTAGCTTTACCCAGGGTCTGCTGCGTCCGCGTGTTGTTAATCAGCTGGGCACCGGCACCGTTTTTCTGAAAGCAGATCAGCTGCCGAGCCTGAAATATATGGATGGTAGCCAGCGTTTTGTGGATGTTCGTTTTAGCCGTTATATGCTGGAAAATGAAATTGGCATGATTTGTGTTCCGCGTGAAAAAAATTGGCTGCGTGAAGTTAGCAGCGGTAGCATGGAAGGTCTGTGGAATACCTTTACCAAAAAATGGCCTCTGGATATCATTAAAGAAACCCAGGCAATTGCCGGTTATAGTAAACTGAATCTGGAACTGGTGTATAATGTGGAAGGTCACCACCACCACCACCACTAAProtein sequence of MBP-KfiA-His₆ (SEQ ID NO: 18) (Note: Italicsections of the sequences are from pMAL-c4X vector and primer.The sequences  for His₆-tag (SEQ ID NO: 22) are underlined)NLGIEGRISEFMIVANMSSYPPRKKELVHSIQSLHAQVDKINLCLNEFEEIPEELDGFSKLNPVIPDKDYKDVGKFIFPCAKNDMIVLTDDDIIYPPDYVEKMLNFYNSFATFNCIVGIHGCIYIDAFDGDQSKRKVESFTQGLLRPRVVNQLGTGTVFLKADQLPSLKYMDGSQRFVDVRFSRYMLENEIGMICVPREKNWLREVSSGSMEGLWNTFTKKWPLDIIKETQAIAGYSKLNLELVYNVE GHHHHHH

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications can be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference.

What is claimed is:
 1. A sialylated oligosaccharide selected from thegroup consisting of: Neu5Acα2-3(Neu5Acα2-6)Galβ1-4Glc;Neu5Acα2-3(Neu5Acα2-6)Galβ1-3GalNAc;Neu5Acα2-3(Neu5Acα2-6)Galβ1-3GlcNAc; andNeu5Acα2-6Galβ1-3GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc.
 2. A sialylatedoligosaccharide which isNeu5Acα2-6Galβ1-4GlcNAcβ1-3(Neu5Acα2-6)Galβ1-4Glc.
 3. A method fortreating necrotizing enterocolitis (NEC), the method comprisingadministering to a subject in need thereof a sialylated oligosaccharideaccording to claim
 1. 4. A method for treating necrotizing enterocolitis(NEC), the method comprising administering to a subject in need thereofthe sialylated oligosaccharide according to claim 2.